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Mitogenomic sequences and evidence from unique gene rearrangements corroborate evolutionary relationships of myctophiformes (Neoteleostei)

Identifieur interne : 000198 ( Pmc/Corpus ); précédent : 000197; suivant : 000199

Mitogenomic sequences and evidence from unique gene rearrangements corroborate evolutionary relationships of myctophiformes (Neoteleostei)

Auteurs : Jan Y. Poulsen ; Ingvar Byrkjedal ; Endre Willassen ; David Rees ; Hirohiko Takeshima ; Takashi P. Satoh ; Gento Shinohara ; Mutsumi Nishida ; Masaki Miya

Source :

RBID : PMC:3682873

Abstract

Background

A skewed assemblage of two epi-, meso- and bathypelagic fish families makes up the order Myctophiformes – the blackchins Neoscopelidae and the lanternfishes Myctophidae. The six rare neoscopelids show few morphological specializations whereas the divergent myctophids have evolved into about 250 species, of which many show massive abundances and wide distributions. In fact, Myctophidae is by far the most abundant fish family in the world, with plausible estimates of more than half of the oceans combined fish biomass. Myctophids possess a unique communication system of species-specific photophore patterns and traditional intrafamilial classification has been established to reflect arrangements of photophores. Myctophids present the most diverse array of larval body forms found in fishes although this attribute has both corroborated and confounded phylogenetic hypotheses based on adult morphology. No molecular phylogeny is available for Myctophiformes, despite their importance within all ocean trophic cycles, open-ocean speciation and as an important part of neoteleost divergence. This study attempts to resolve major myctophiform phylogenies from both mitogenomic sequences and corroborating evidence in the form of unique mitochondrial gene order rearrangements.

Results

Mitogenomic evidence from DNA sequences and unique gene orders are highly congruent concerning phylogenetic resolution on several myctophiform classification levels, corroborating evidence from osteology, larval ontogeny and photophore patterns, although the lack of larval morphological characters within the subfamily Lampanyctinae stands out. Neoscopelidae is resolved as the sister family to myctophids with Solivomer arenidens positioned as a sister taxon to the remaining neoscopelids. The enigmatic Notolychnus valdiviae is placed as a sister taxon to all other myctophids and exhibits an unusual second copy of the tRNA-Met gene – a gene order rearrangement reminiscent of that found in the tribe Diaphini although our analyses show it to be independently derived. Most tribes are resolved in accordance with adult morphology although Gonichthyini is found within a subclade of the tribe Myctophini consisting of ctenoid scaled species. Mitogenomic sequence data from this study recognize 10 reciprocally monophyletic lineages within Myctophidae, with five of these clades delimited from additional rearranged gene orders or intergenic non-coding sequences.

Conclusions

Mitogenomic results from DNA sequences and unique gene orders corroborate morphology in phylogeny reconstruction and provide a likely scenario for the phylogenetic history of Myctophiformes. The extent of gene order rearrangements found within the mitochondrial genomes of myctophids is unique for phylogenetic purposes.


Url:
DOI: 10.1186/1471-2148-13-111
PubMed: 23731841
PubMed Central: 3682873

Links to Exploration step

PMC:3682873

Le document en format XML

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<name sortKey="Willassen, Endre" sort="Willassen, Endre" uniqKey="Willassen E" first="Endre" last="Willassen">Endre Willassen</name>
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<title>Background</title>
<p>A skewed assemblage of two epi-, meso- and bathypelagic fish families makes up the order Myctophiformes – the blackchins Neoscopelidae and the lanternfishes Myctophidae. The six rare neoscopelids show few morphological specializations whereas the divergent myctophids have evolved into about 250 species, of which many show massive abundances and wide distributions. In fact, Myctophidae is by far the most abundant fish family in the world, with plausible estimates of more than half of the oceans combined fish biomass. Myctophids possess a unique communication system of species-specific photophore patterns and traditional intrafamilial classification has been established to reflect arrangements of photophores. Myctophids present the most diverse array of larval body forms found in fishes although this attribute has both corroborated and confounded phylogenetic hypotheses based on adult morphology. No molecular phylogeny is available for Myctophiformes, despite their importance within all ocean trophic cycles, open-ocean speciation and as an important part of neoteleost divergence. This study attempts to resolve major myctophiform phylogenies from both mitogenomic sequences and corroborating evidence in the form of unique mitochondrial gene order rearrangements.</p>
</sec>
<sec>
<title>Results</title>
<p>Mitogenomic evidence from DNA sequences and unique gene orders are highly congruent concerning phylogenetic resolution on several myctophiform classification levels, corroborating evidence from osteology, larval ontogeny and photophore patterns, although the lack of larval morphological characters within the subfamily Lampanyctinae stands out. Neoscopelidae is resolved as the sister family to myctophids with
<italic>Solivomer arenidens</italic>
positioned as a sister taxon to the remaining neoscopelids. The enigmatic
<italic>Notolychnus valdiviae</italic>
is placed as a sister taxon to all other myctophids and exhibits an unusual second copy of the tRNA-Met gene – a gene order rearrangement reminiscent of that found in the tribe Diaphini although our analyses show it to be independently derived. Most tribes are resolved in accordance with adult morphology although Gonichthyini is found within a subclade of the tribe Myctophini consisting of ctenoid scaled species. Mitogenomic sequence data from this study recognize 10 reciprocally monophyletic lineages within Myctophidae, with five of these clades delimited from additional rearranged gene orders or intergenic non-coding sequences.</p>
</sec>
<sec>
<title>Conclusions</title>
<p>Mitogenomic results from DNA sequences and unique gene orders corroborate morphology in phylogeny reconstruction and provide a likely scenario for the phylogenetic history of Myctophiformes. The extent of gene order rearrangements found within the mitochondrial genomes of myctophids is unique for phylogenetic purposes.</p>
</sec>
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</TEI>
<pmc article-type="research-article" xml:lang="en">
<pmc-dir>properties open_access</pmc-dir>
<front>
<journal-meta>
<journal-id journal-id-type="nlm-ta">BMC Evol Biol</journal-id>
<journal-id journal-id-type="iso-abbrev">BMC Evol. Biol</journal-id>
<journal-title-group>
<journal-title>BMC Evolutionary Biology</journal-title>
</journal-title-group>
<issn pub-type="epub">1471-2148</issn>
<publisher>
<publisher-name>BioMed Central</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="pmid">23731841</article-id>
<article-id pub-id-type="pmc">3682873</article-id>
<article-id pub-id-type="publisher-id">1471-2148-13-111</article-id>
<article-id pub-id-type="doi">10.1186/1471-2148-13-111</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Research Article</subject>
</subj-group>
</article-categories>
<title-group>
<article-title>Mitogenomic sequences and evidence from unique gene rearrangements corroborate evolutionary relationships of myctophiformes (Neoteleostei)</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author" corresp="yes" id="A1">
<name>
<surname>Poulsen</surname>
<given-names>Jan Y</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>Jan.Poulsen@bm.uib.no</email>
</contrib>
<contrib contrib-type="author" id="A2">
<name>
<surname>Byrkjedal</surname>
<given-names>Ingvar</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>Ingvar.Byrkjedal@bm.uib.no</email>
</contrib>
<contrib contrib-type="author" id="A3">
<name>
<surname>Willassen</surname>
<given-names>Endre</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>Endre.Willassen@bm.uib.no</email>
</contrib>
<contrib contrib-type="author" id="A4">
<name>
<surname>Rees</surname>
<given-names>David</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>David.Rees@bm.uib.no</email>
</contrib>
<contrib contrib-type="author" id="A5">
<name>
<surname>Takeshima</surname>
<given-names>Hirohiko</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<email>takeshim@aori.u-tokyo.ac.jp</email>
</contrib>
<contrib contrib-type="author" id="A6">
<name>
<surname>Satoh</surname>
<given-names>Takashi P</given-names>
</name>
<xref ref-type="aff" rid="I3">3</xref>
<email>satotaka@kahaku.go.jp</email>
</contrib>
<contrib contrib-type="author" id="A7">
<name>
<surname>Shinohara</surname>
<given-names>Gento</given-names>
</name>
<xref ref-type="aff" rid="I3">3</xref>
<email>s-gento@kahaku.go.jp</email>
</contrib>
<contrib contrib-type="author" id="A8">
<name>
<surname>Nishida</surname>
<given-names>Mutsumi</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<email>mnishida@aori.u-tokyo.ac.jp</email>
</contrib>
<contrib contrib-type="author" id="A9">
<name>
<surname>Miya</surname>
<given-names>Masaki</given-names>
</name>
<xref ref-type="aff" rid="I4">4</xref>
<email>miya@chiba-muse.or.jp</email>
</contrib>
</contrib-group>
<aff id="I1">
<label>1</label>
Natural History Collections, University Museum of Bergen, University of Bergen, Allégaten 41, P.O. Box 7800, Bergen N-5020, Norway</aff>
<aff id="I2">
<label>2</label>
Department of Marine Bioscience, Atmosphere and Ocean Research Institute, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8564, Japan</aff>
<aff id="I3">
<label>3</label>
National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan</aff>
<aff id="I4">
<label>4</label>
Department of Zoology, Natural History Museum and Institute, 955-2 Aoba-cho, Chuo-ku, Chiba 260-8682, Japan</aff>
<pub-date pub-type="collection">
<year>2013</year>
</pub-date>
<pub-date pub-type="epub">
<day>3</day>
<month>6</month>
<year>2013</year>
</pub-date>
<volume>13</volume>
<fpage>111</fpage>
<lpage>111</lpage>
<history>
<date date-type="received">
<day>17</day>
<month>2</month>
<year>2012</year>
</date>
<date date-type="accepted">
<day>20</day>
<month>5</month>
<year>2013</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright © 2013 Poulsen et al.; licensee BioMed Central Ltd.</copyright-statement>
<copyright-year>2013</copyright-year>
<copyright-holder>Poulsen et al.; licensee BioMed Central Ltd.</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0">
<license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0">http://creativecommons.org/licenses/by/2.0</ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
</license>
</permissions>
<self-uri xlink:href="http://www.biomedcentral.com/1471-2148/13/111"></self-uri>
<abstract>
<sec>
<title>Background</title>
<p>A skewed assemblage of two epi-, meso- and bathypelagic fish families makes up the order Myctophiformes – the blackchins Neoscopelidae and the lanternfishes Myctophidae. The six rare neoscopelids show few morphological specializations whereas the divergent myctophids have evolved into about 250 species, of which many show massive abundances and wide distributions. In fact, Myctophidae is by far the most abundant fish family in the world, with plausible estimates of more than half of the oceans combined fish biomass. Myctophids possess a unique communication system of species-specific photophore patterns and traditional intrafamilial classification has been established to reflect arrangements of photophores. Myctophids present the most diverse array of larval body forms found in fishes although this attribute has both corroborated and confounded phylogenetic hypotheses based on adult morphology. No molecular phylogeny is available for Myctophiformes, despite their importance within all ocean trophic cycles, open-ocean speciation and as an important part of neoteleost divergence. This study attempts to resolve major myctophiform phylogenies from both mitogenomic sequences and corroborating evidence in the form of unique mitochondrial gene order rearrangements.</p>
</sec>
<sec>
<title>Results</title>
<p>Mitogenomic evidence from DNA sequences and unique gene orders are highly congruent concerning phylogenetic resolution on several myctophiform classification levels, corroborating evidence from osteology, larval ontogeny and photophore patterns, although the lack of larval morphological characters within the subfamily Lampanyctinae stands out. Neoscopelidae is resolved as the sister family to myctophids with
<italic>Solivomer arenidens</italic>
positioned as a sister taxon to the remaining neoscopelids. The enigmatic
<italic>Notolychnus valdiviae</italic>
is placed as a sister taxon to all other myctophids and exhibits an unusual second copy of the tRNA-Met gene – a gene order rearrangement reminiscent of that found in the tribe Diaphini although our analyses show it to be independently derived. Most tribes are resolved in accordance with adult morphology although Gonichthyini is found within a subclade of the tribe Myctophini consisting of ctenoid scaled species. Mitogenomic sequence data from this study recognize 10 reciprocally monophyletic lineages within Myctophidae, with five of these clades delimited from additional rearranged gene orders or intergenic non-coding sequences.</p>
</sec>
<sec>
<title>Conclusions</title>
<p>Mitogenomic results from DNA sequences and unique gene orders corroborate morphology in phylogeny reconstruction and provide a likely scenario for the phylogenetic history of Myctophiformes. The extent of gene order rearrangements found within the mitochondrial genomes of myctophids is unique for phylogenetic purposes.</p>
</sec>
</abstract>
<kwd-group>
<kwd>Myctophiformes</kwd>
<kwd>Myctophidae</kwd>
<kwd>Neoscopelidae</kwd>
<kwd>Phylogeny</kwd>
<kwd>Mitogenomics</kwd>
<kwd>Gene rearrangements</kwd>
<kwd>Non-coding sequence</kwd>
</kwd-group>
</article-meta>
</front>
<body>
<sec>
<title>Background</title>
<p>Lanternfishes (Myctophidae) and blackchins (Neoscopelidae) comprise the only two families in the order Myctophiformes, superorder Scopelomorpha [
<xref ref-type="bibr" rid="B1">1</xref>
], a group currently including some 250 species [
<xref ref-type="bibr" rid="B2">2</xref>
]. The 32 genera of Myctophidae contain about 98% of the species diversity, and morphological characters have clearly shown Myctophidae more derived compared to the more generalized Neoscopelidae [
<xref ref-type="bibr" rid="B3">3</xref>
-
<xref ref-type="bibr" rid="B6">6</xref>
]. Myctophids are exclusively marine and pelagic, occupying depths from surface waters down to the upper 1000 meters of the bathypelagic layer (1000–4000 meters). The group as a whole constitutes fairly small fishes ranging in size from 20 to 300 millimeters, with larger members being confined to the bathypelagic realm. They are ubiquitous in the World’s oceans and exhibit extremely high abundances with one estimate as high as 65% of all pelagic deep-sea fish biomass [
<xref ref-type="bibr" rid="B7">7</xref>
].</p>
<p>The most striking feature of myctophids is their often species-specific patterns of photophores (light-organs), which have earned them their popular name lanternfishes. Photophores are named according to specific patterns present in various degrees throughout different myctophid lineages (Figure 
<xref ref-type="fig" rid="F1">1</xref>
), making photophore patterns important when reconstructing the evolution of myctophids. It is generally believed that species-specific flash communication underpins the evolution of photophore patterns although myctophids are also macrosmatic, i.e. possessing large olfactory organs, indicating a complicated communication system not fully understood [
<xref ref-type="bibr" rid="B8">8</xref>
]. Myctophid fishes are believed to communicate in a firefly-like manner by using duration- and intensity-variable flashes of light produced by species-specific and sexually dimorphic patterned photophores [
<xref ref-type="bibr" rid="B3">3</xref>
,
<xref ref-type="bibr" rid="B9">9</xref>
]. One argument against flash communication is the almost identical photophore patterns observed between some species, making it virtually impossible to discriminate between flashes except within very close range [
<xref ref-type="bibr" rid="B10">10</xref>
]. Although bioluminescence has evolved multiple times across open-ocean organisms [
<xref ref-type="bibr" rid="B11">11</xref>
], the possibility of using this feature in tracing the natural history of an entire clade is, however, unique within open-ocean and deep-sea animals. The mechanism behind myctophid bioluminescence has caused some debate since bioluminescent bacteria of the strain
<italic>Vibrio</italic>
were found by Foran [
<xref ref-type="bibr" rid="B12">12</xref>
] using hybridization probes and later challenged by Haygood et al. [
<xref ref-type="bibr" rid="B13">13</xref>
] who confidently excluded both symbiotic bacteria and bacterial luciferase as the light source. It is now generally accepted that a coelenterazine system is responsible for myctophid bioluminescence [
<xref ref-type="bibr" rid="B11">11</xref>
,
<xref ref-type="bibr" rid="B14">14</xref>
], contrary to e.g. the famous deep-sea anglerfishes that possess family-specific symbiotic photobacterial strains of
<italic>Vibrio</italic>
in the esca [
<xref ref-type="bibr" rid="B15">15</xref>
,
<xref ref-type="bibr" rid="B16">16</xref>
]. Most myctophids that have been examined concerning their visual spectrum show a tendency towards longer wavelengths for absorption and emission, resulting in a dim blue bioluminescence as typically found in deep-sea fishes [
<xref ref-type="bibr" rid="B17">17</xref>
]. The latter study found a relatively confined spectral range of absorption in myctophids compared to other mesopelagic fishes. However, Hasegawa et al. [
<xref ref-type="bibr" rid="B18">18</xref>
] found that
<italic>Myctophum nitidulum</italic>
also possessed longer wavelength retinal pigments enabling them to detect a broader spectrum of wavelengths produced by other animals, and/or their own light emission and down-welling sunlight. Extended spectral range is familiar from loose-jaw dragonfishes well-known for emitting long-wavelength red light from orbital photophores, enabling them to communicate or detect prey at wavelengths invisible to other deep-sea organisms [
<xref ref-type="bibr" rid="B19">19</xref>
]. Turner et al. [
<xref ref-type="bibr" rid="B17">17</xref>
] note that possession of a relatively limited spectral range could be the result of a shared evolutionary history more than multiple instances of visual adaptation to the mesopelagic environment.</p>
<fig id="F1" position="float">
<label>Figure 1</label>
<caption>
<p>
<bold>
<italic>Benthosema glaciale, </italic>
</bold>
<bold>© Rudolf Svendsen, Høgsfjorden, Norway.</bold>
Standard myctophid photophore terminology noted with photophores/luminescent organs truncated if found in other myctophids than
<italic>B. glaciale</italic>
. Abbreviations: Ant – Anterior orbital (antorbital), AOa – Anal organs anterior, AOp – Anal organs posterior, Dn – Dorsal nasal (dorsonasal), InGl – Infracaudal gland, LO – Luminous organ, PO – Pectoral organs, Pol – Posterior organ lateral, PLO – Pectoral lateral organ, Prc – Precaudal organs (precaudals), PVO – Pectoral ventral organs, Sao – Supraanal organs, So – Suborbital, Gl – Supracaudal gland, VLO – Ventral lateral organ, Vn – Ventral nasal (ventronasal), VO – Ventral organs.</p>
</caption>
<graphic xlink:href="1471-2148-13-111-1"></graphic>
</fig>
<p>Diel vertical migration (DVM) is a prominent feature of myctophids although not all myctophid species engage in this activity [
<xref ref-type="bibr" rid="B20">20</xref>
]. DVM prompted the famous “false bottom” (or the deep scattering layer) discovery, turning out to be massive occurrences of the swimbladder-possessing myctophid
<italic>Ceratoscopelus maderensis</italic>
among other deep-sea animals [
<xref ref-type="bibr" rid="B21">21</xref>
]. DVM is common across many animal groups and is generally believed to involve feeding and/or reduced predation risk associated with reduced light intensity [
<xref ref-type="bibr" rid="B22">22</xref>
]. Herring [
<xref ref-type="bibr" rid="B23">23</xref>
] notes that if populations are biased towards horizontal layers, as is often the case with myctophids, DVM might be an effective way to channel intra-specific encounters and could be important in mate recognition. A phylogenetic component in DVM has not been explored although DVM is clearly more pronounced in some groups of myctophids than others, e.g. the slendertails of the tribe Gonichthyini including the genera
<italic>Gonichthys, Centrobranchus, Tarletonbeania</italic>
and
<italic>Loweina</italic>
.</p>
<p>Stiassny [
<xref ref-type="bibr" rid="B5">5</xref>
] and Yamaguchi [
<xref ref-type="bibr" rid="B24">24</xref>
] have most recently reviewed myctophiform synapomorphies and there is little doubt that the order constitutes a monophyletic assemblage. Intrarelationships of Neoscopelidae (3 genera and 6 species) are not well resolved [
<xref ref-type="bibr" rid="B5">5</xref>
] with generic relationships within Myctophidae better understood bearing on the classification initially by Bolin [
<xref ref-type="bibr" rid="B6">6</xref>
], subsequently Fraser-Brunner [
<xref ref-type="bibr" rid="B4">4</xref>
], and finally Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
]. They classified the order into two families, divided the myctophids into two subfamilies that together include six tribes, based on osteology and photophore patterns (Figure 
<xref ref-type="fig" rid="F2">2</xref>
A). This classification is still in use today, except for the lineage Electronini also being recognized as a distinct tribe as originally designated by Wisner [
<xref ref-type="bibr" rid="B25">25</xref>
]. Additional corroborating evidence for this classification has been presented partly from urodermal bones [
<xref ref-type="bibr" rid="B26">26</xref>
] and notably a broad range of larval body forms [
<xref ref-type="bibr" rid="B27">27</xref>
-
<xref ref-type="bibr" rid="B30">30</xref>
] (Figure 
<xref ref-type="fig" rid="F2">2</xref>
B-D). Myctophid larvae show some of the most diverse forms within any teleost order and characters associated with early ontogeny have proven valuable in phylogenetics [
<xref ref-type="bibr" rid="B30">30</xref>
]. Stiassny [
<xref ref-type="bibr" rid="B5">5</xref>
] added new data and reviewed existing characters, resulting in a topology little resolved below subfamily level (Figure 
<xref ref-type="fig" rid="F2">2</xref>
E). Yamaguchi [
<xref ref-type="bibr" rid="B24">24</xref>
] carried out the most recent analysis using the characters provided by the previous studies, which again resulted in an unresolved topology (Figure 
<xref ref-type="fig" rid="F2">2</xref>
F). Morphological work on photophore patterns, adult osteology and larval ontogeny have clearly established their utility in myctophid phylogenetics, although missing larval characters in the subfamily Lampanyctinae have been a significant source of discrepancy between morphological hypotheses [
<xref ref-type="bibr" rid="B29">29</xref>
]. Larval characters corroborate parts of adult phylogenetic reconstruction (Figure 
<xref ref-type="fig" rid="F2">2</xref>
) although the larvae of the large genus
<italic>Diaphus</italic>
have proven exceptionally difficult to identify [
<xref ref-type="bibr" rid="B30">30</xref>
]. The most persistent issue concerning myctophid phylogenetics has been the placement of
<italic>Notolychnus valdiviae</italic>
, a monotypic, diminuitive, putatively plesiomorphic myctophid showing dorsally located photophores, weak ossification and several characters difficult to assess even on subfamily level [
<xref ref-type="bibr" rid="B3">3</xref>
]. Recent cladistic analyses of morphological characters have converged on
<italic>Notolychnus</italic>
as sister taxon to the remaining lampanyctines (Figure 
<xref ref-type="fig" rid="F2">2</xref>
) as initially suggested by Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
].</p>
<fig id="F2" position="float">
<label>Figure 2</label>
<caption>
<p>
<bold>Phylogenetic hypotheses from previous morphological studies. A</bold>
) Adult osteology [
<xref ref-type="bibr" rid="B3">3</xref>
]. Parenthesized genera no longer recognized.
<bold>B</bold>
) Larval development (Myctophinae only) with topology constructed from written considerations by Moser & Ahlstrom [
<xref ref-type="bibr" rid="B27">27</xref>
] and tribes inferred according to [
<xref ref-type="bibr" rid="B3">3</xref>
].
<bold>C</bold>
) Larval development (Lampanyctinae only) [
<xref ref-type="bibr" rid="B28">28</xref>
].
<bold>D</bold>
) Adult osteology + larval development [
<xref ref-type="bibr" rid="B30">30</xref>
].
<bold>E</bold>
) Unordered multistate parsimony analysis of adult + larval characters including 4 new characters [
<xref ref-type="bibr" rid="B5">5</xref>
].
<bold>F</bold>
) Ordered multistate parsimony reanalysis of all adult and larval characters [
<xref ref-type="bibr" rid="B24">24</xref>
].</p>
</caption>
<graphic xlink:href="1471-2148-13-111-2"></graphic>
</fig>
<p>Surprisingly little molecular work has been carried out on Myctophiformes considering their abundance and at times problematic species identification. Yamaguchi et al. [
<xref ref-type="bibr" rid="B31">31</xref>
] tested photophore patterns, biogeographical distributions and diverse larval eye morphology within
<italic>Hygophum</italic>
in a phylogenetic context. The results indicated all three attributes as somewhat informative concerning speciation pattern, although clear conclusions were hard to extract due to few characters (partial 16S rRNA gene sequences) and incomplete taxon sampling. However, with an increasing understanding of myctophiform phylogeny, this type of study will become highly valuable in terms of assessing open-ocean speciation patterns in myctophids as well as other open-ocean fish components [
<xref ref-type="bibr" rid="B32">32</xref>
].</p>
<p>Miya et al. [
<xref ref-type="bibr" rid="B33">33</xref>
] produced the first mitochondrial (mt) genome mapping of myctophiform fishes, showing that the two myctophids
<italic>Diaphus splendidus</italic>
and
<italic>Myctophum affine</italic>
possess non-typical vertebrate mt gene arrangements, whereas the neoscopelid fish
<italic>Neoscopelus microchir</italic>
exhibits the typical gene arrangement for vertebrates [
<xref ref-type="bibr" rid="B34">34</xref>
]. Their findings indicated that mt gene arrangements of myctophid fishes have the potential to provide evidence concerning clade delimitation at various levels. Clade-specific gene orders are considered strong evidence for monophyly since the process of gene duplication and subsequent deletions are considered random and rare events [
<xref ref-type="bibr" rid="B35">35</xref>
-
<xref ref-type="bibr" rid="B39">39</xref>
]. However, accumulating evidence shows positive selection on some rearranged mt gene orders [
<xref ref-type="bibr" rid="B40">40</xref>
] to be much more common than initially expected [
<xref ref-type="bibr" rid="B41">41</xref>
]. Intergenic non-coding (INC) regions are likewise believed to provide evidence for phylogenetic history, although this group of markers has been poorly explored mainly because of insufficient taxon sampling.</p>
<p>This study is the first to use mitogenomic DNA sequences combined with additional information from extensively rearranged gene orders and INC-sequences to explore myctophiform phylogeny.</p>
</sec>
<sec sec-type="methods">
<title>Methods</title>
<sec>
<title>Taxon sampling</title>
<p>Twenty-three genera and a total of 41 species of myctophiform fishes are included in this study – 38 species representing new data and three from a previous mitogenomic study on higher teleostean phylogeny [
<xref ref-type="bibr" rid="B33">33</xref>
]. Forty-six outgroup taxa ranging from early euteleost groups (root) to ophidiiform and lophiiform fishes (Percomorpha) were chosen with all putative myctophiform sister groups included. Taxonomic sampling focused on tribal classification sensu Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] representing the distinct myctophiform lineages. We sampled all non-monotypic tribes with multiple genera although only a single representative of Gonichthyini is included due to technical difficulties in amplification of DNA. A large number of taxa within the genus
<italic>Diaphus</italic>
were included since about 1/3 of all myctophid species are placed within this genus. Voucher specimens were obtained from sampling onboard Research Vessel Pâmiut (Greenland Institute of Natural Resources) around Greenland (JYP), from the MAR-ECO cruise (IB), catalogued and uncatalogued material at NSMT (TPS and GS) and institutional loans from ASIZP, BSKU, KU, MCZ, SIO, WU and ZMUC (Table 
<xref ref-type="table" rid="T1">1</xref>
). Voucher specimens were examined and photographed with e-vouchers available online [
<xref ref-type="bibr" rid="B42">42</xref>
].</p>
<table-wrap position="float" id="T1">
<label>Table 1</label>
<caption>
<p>87 taxa included in this study</p>
</caption>
<table frame="hsides" rules="groups" border="1">
<colgroup>
<col align="left"></col>
<col align="left"></col>
<col align="left"></col>
<col align="left"></col>
<col align="left"></col>
<col align="left"></col>
</colgroup>
<thead valign="top">
<tr>
<th align="left">
<bold>Clade</bold>
</th>
<th align="left">
<bold>Species</bold>
</th>
<th align="left">
<bold>Acc. #</bold>
</th>
<th align="left">
<bold>Tissue #</bold>
</th>
<th align="left">
<bold>Voucher Museum #</bold>
</th>
<th align="left">
<bold>Study</bold>
</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="left" valign="bottom">
<bold>Esociformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Esox lucius</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004103
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Ishiguro et al. 2003 [
<xref ref-type="bibr" rid="B43">43</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Dallia pectoralis</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004102
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Ishiguro et al. 2003 [
<xref ref-type="bibr" rid="B43">43</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Salmoniformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Coregonus lavaretus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB034824
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya & Nishida 2000 [
<xref ref-type="bibr" rid="B44">44</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Thymallus thymallus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">FJ853655
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Yasuike et al. 2010
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Salvelinus alpinus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AF154851
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Doiron et al. 2002
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Argentiniformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Bathylagus ochotensis</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004101
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Ishiguro et al. 2003 [
<xref ref-type="bibr" rid="B43">43</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Nansenia ardesiaca</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004106
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Ishiguro et al. 2003 [
<xref ref-type="bibr" rid="B43">43</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Glossanodon semifasciatus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004105
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Ishiguro et al. 2003 [
<xref ref-type="bibr" rid="B43">43</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Opistophroctus soleatus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004110
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Ishiguro et al. 2003 [
<xref ref-type="bibr" rid="B43">43</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Osmeriformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Retropinna retropinna</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004108
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Ishiguro et al. 2003 [
<xref ref-type="bibr" rid="B43">43</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Salangichthys microdon</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004109
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Ishiguro et al. 2003 [
<xref ref-type="bibr" rid="B43">43</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Mallotus villosus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">HM106491
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Li et al. 2010
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Plecoglossus altivelis</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB047553
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Ishiguro et al. 2001
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Stomiiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Diplophos taenia</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB034825
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya & Nishida 2000 [
<xref ref-type="bibr" rid="B44">44</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Chauliodus sloani</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002915
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Gonostoma gracile</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB016274
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya & Nishida 1999 [
<xref ref-type="bibr" rid="B45">45</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Galaxiiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Galaxiella nigrostriata</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP006853
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. (unpubl.)
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Galaxias maculates</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004104
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Ishiguro et al. 2003 [
<xref ref-type="bibr" rid="B43">43</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Galaxias gollumoides</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">HM106487
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Li et al. 2010
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Aulopiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Synodus variegatus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AY524977
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Chen & Wu (unpubl.)
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Chlorophthalmus agassizi</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002918
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Aulopus japonicus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB047821
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Kawaguchi et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Harpadon microchir</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002919
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Saurida undosquamis</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002920
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Ateleopodiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Ateleopus japonicus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002916
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Ijimaia dofleini</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002917
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Lampriformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Lampris guttatus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002924
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Zu cristatus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002926
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Trachipterus trachypterus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002925
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Myctophiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Neoscopelus macrolepidotus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012238
<hr></hr>
</td>
<td align="left" valign="bottom">KU 3297
<hr></hr>
</td>
<td align="left" valign="bottom">Uncatalogued
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Neoscopelus microchir</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002921
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Scopelengys tristis</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012228
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 99997
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 99997
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Solivomer arenidens</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012249
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT
<hr></hr>
</td>
<td align="left" valign="bottom">Uncatalogued
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Benthosema fibulatum</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012253
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 75816
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 75816
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Benthosema glaciale</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012264
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC #8160
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC P2393965
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Benthosema pterotum</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012260
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 75855
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 75855
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Bolinichthys distofax</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012232
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 97624
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 97624
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Bolinichthys pyrsobolus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012261
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 92273
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 92273
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Centrobranchus choerocephalus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012237
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 102902
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 102902
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Ceratoscopelus maderensis</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012259
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC #6600
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC P2393929
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Diaphus chrysorhynchus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012230
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 79902
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 79902
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Diaphus gigas</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012235
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 91530
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 91530
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Diaphus luetkeni</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012231
<hr></hr>
</td>
<td align="left" valign="bottom">ASIZP 911514
<hr></hr>
</td>
<td align="left" valign="bottom">ASIZP66276
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Diaphus splendidus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002923
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Diaphus theta</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012240
<hr></hr>
</td>
<td align="left" valign="bottom">KU 2135
<hr></hr>
</td>
<td align="left" valign="bottom">KU 27971
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Diogenichthys atlanticus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012233
<hr></hr>
</td>
<td align="left" valign="bottom">SIO 09320
<hr></hr>
</td>
<td align="left" valign="bottom">SIO 09320
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Electrona antarctica</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012248
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC #7552
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC P2393962
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Gymnoscopelus nicholsi</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012250
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC #7806
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC P2393963
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Krefftichthys anderssoni</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012236
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC #7550
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC P2393964
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Lampadena anomala</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012227
<hr></hr>
</td>
<td align="left" valign="bottom">ME 4201#3
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUB 18019
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Lampadena urophaos atlantica</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012251
<hr></hr>
</td>
<td align="left" valign="bottom">ME 7371#4
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUB 18050
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Lampadena yaquinae</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012257
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 72341
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 72341
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Lampanyctus macdonaldi</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012241
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC #8077
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC P2393967
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Lampanyctus crocodilus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012258
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC #8076
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC P2393968
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Lobianchia gemellarii</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012242
<hr></hr>
</td>
<td align="left" valign="bottom">ME 4239#6
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUB O.1725
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Myctophum affine</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002922
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Myctophum asperum</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012234
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 91490
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 91490
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Myctophum nitidulum</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012255
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 92345
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 92345
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Myctophum orientale</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012254
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 77350
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 77350
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Myctophum punctatum</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012239
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC #6594
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC P2393969
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Nannobrachium ritteri</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012247
<hr></hr>
</td>
<td align="left" valign="bottom">KU 2237
<hr></hr>
</td>
<td align="left" valign="bottom">KU 28276
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Notoscopelus caudispinosus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012256
<hr></hr>
</td>
<td align="left" valign="bottom">KU 5301
<hr></hr>
</td>
<td align="left" valign="bottom">MCZ 161883
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Notoscopelus japonicus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012252
<hr></hr>
</td>
<td align="left" valign="bottom">BSKU 103772
<hr></hr>
</td>
<td align="left" valign="bottom">BSKU 103772
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Notoscopelus kroyeri</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012262
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC #8078
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC P2393970
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Notolychnus valdiviae</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012229
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 102930
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 102930
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Protomyctophum arcticum</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB648909-10
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC #8476
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUC P2393971
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Stenobrachius leucopsarus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012245
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 78748
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 78748
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Symbolophorus californiensis</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012246
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 92257
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 92257
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Taaningichthys minimus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012244
<hr></hr>
</td>
<td align="left" valign="bottom">ME 5633
<hr></hr>
</td>
<td align="left" valign="bottom">ZMUB 18049
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Triphoturus nigrescens</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP012243
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 102934
<hr></hr>
</td>
<td align="left" valign="bottom">NSMT-P 102934
<hr></hr>
</td>
<td align="left" valign="bottom">This study
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Ophidiiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Cataetyx rubrirostris</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004407
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2003 [
<xref ref-type="bibr" rid="B46">46</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Carapus bermudensis</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004404
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2003 [
<xref ref-type="bibr" rid="B46">46</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Bassozetus zenkevitchi</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004405
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2003 [
<xref ref-type="bibr" rid="B46">46</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Lophiiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Sladenia gardineri</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB282827
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2010 [
<xref ref-type="bibr" rid="B47">47</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Coelophrys brevicaudata</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB282834
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2010 [
<xref ref-type="bibr" rid="B47">47</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Thaumatichthys pagidostomus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB282847
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2010 [
<xref ref-type="bibr" rid="B47">47</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Percopsiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Percopsis transmontana</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002928
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Aphredoderus sayanus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004403
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2003 [
<xref ref-type="bibr" rid="B46">46</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Polymixiiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Polymixia japonica</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB034826
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya & Nishida 2000 [
<xref ref-type="bibr" rid="B44">44</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Polymixia lowei</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002927
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Gadiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Stylephorus chordatus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AB280688
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2007
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Bregmaceros transmontana</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004411
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2003 [
<xref ref-type="bibr" rid="B46">46</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Trachyrincus murrayi</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP008990
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Satoh et al. 2006 [
<xref ref-type="bibr" rid="B48">48</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Lota lota</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004412
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2003 [
<xref ref-type="bibr" rid="B46">46</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">
<bold>Zeiformes</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Zeus faber</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP002941
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2001 [
<xref ref-type="bibr" rid="B33">33</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">
<bold>
<italic>Parazen pacificus</italic>
</bold>
<hr></hr>
</td>
<td align="left" valign="bottom">AP004433
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom"> 
<hr></hr>
</td>
<td align="left" valign="bottom">Miya et al. 2003 [
<xref ref-type="bibr" rid="B46">46</xref>
]
<hr></hr>
</td>
</tr>
<tr>
<td align="left"> </td>
<td align="left">
<bold>
<italic>Zenion japonicum</italic>
</bold>
</td>
<td align="left">AP004434</td>
<td align="left"> </td>
<td align="left"> </td>
<td align="left">Miya et al. 2003 [
<xref ref-type="bibr" rid="B46">46</xref>
]</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<p>Taxa included in this study listed according to ordinal classification. Mitogenome accession number, tissue number, specimen + institute number and study noted if information available.</p>
</table-wrap-foot>
</table-wrap>
</sec>
<sec>
<title>Production of mitogenomic data</title>
<p>Genomic DNA was extracted using the Qiagen Puregene extraction kit following the manufacturer´s protocol and used directly for long and accurate amplification PCR (LA PCR) of the entire mitochondrial genome [
<xref ref-type="bibr" rid="B49">49</xref>
]. Depending on tissue quality and LA primer fidelity, a variable number of LA PCR reactions were needed for complete coverage, with a minimum of two and a maximum of six for some species. The LA PCR product was diluted with ddH
<sub>2</sub>
O according to success of amplification and nested short PCR was subsequently performed on the LA product with a broad range of fish universal and species specific primers (>200). Double stranded PCR products were cleaned with Exo-Sap at 60°C for 60 minutes and used as template for direct cycle-sequencing with dye labeled terminators (Applied Biosystems). All fragments were sequenced on automated DNA sequencers according to length of labelled fragments. Quality of tissue was shown to be a prominent obstacle regarding successful amplifications. Issues regarding amplification necessitated multiple PCR protocols and tailor-made primer sets for different taxa. Variable number tandem repeats found in the control region and various INC-regions added to the technical issues. Primer information and protocols regarding PCR and sequencing for particular species are available by request (JYP). Newly determined mitogenome sequences are available as [DDBJ, EMBL or GenBank AP012227-62, AP0122264 and AB648909-10] (Table 
<xref ref-type="table" rid="T1">1</xref>
).</p>
</sec>
<sec>
<title>Sequence editing, alignment and analyses</title>
<p>Raw sequence data were processed and formatted with the software 4peaks Ver. 1.7.2 [
<xref ref-type="bibr" rid="B50">50</xref>
], Textwrangler Ver. 3.5.3 [
<xref ref-type="bibr" rid="B51">51</xref>
] and MacClade Ver. 4.06 [
<xref ref-type="bibr" rid="B52">52</xref>
] at various stages before alignment. Mitogenome assembly was performed using Sequencher Ver. 4.10.1 [
<xref ref-type="bibr" rid="B53">53</xref>
] by concatenation of overlapping sequences. Numerous single nucleotide repeat regions were concatenated from the L- and H-strand sequences assuming the repeat region to be identical in the two strands. This was verified by electrophoresis of the sequenced PCR products showing bands with equal size to the sequenced fragments. All genes were initially annotated by alignment to those from closely related fishes already available in GenBank [
<xref ref-type="bibr" rid="B54">54</xref>
]. Subsequently, protein coding genes were examined by reference to ORFs and the 22 tRNA genes were examined for secondary structure by tRNAscan-SE Ver. 1.21 [
<xref ref-type="bibr" rid="B55">55</xref>
,
<xref ref-type="bibr" rid="B56">56</xref>
]. The two ribosomal RNA genes (12S and 16S) were annotated as the sequences between flanking tRNA Phe, Val and Leu
<sup>UUR</sup>
in addition to alignment using previously determined myctophid RNA genes [
<xref ref-type="bibr" rid="B33">33</xref>
].</p>
<p>Alignment of the 12 protein coding genes was done by eye whereas the 22 tRNA genes and 12S+16S rRNA genes were aligned with a probabilistic multiple alignment approach using ProAlign Ver. 0.5 [
<xref ref-type="bibr" rid="B57">57</xref>
]. The ND6 gene was excluded due to heterogeneous base composition [
<xref ref-type="bibr" rid="B44">44</xref>
] leaving 10908 protein coding nucleotide characters (3636 amino acids). Ambiguous alignment of the tRNA and rRNA genes were discarded from the analyses based on the 90% minimum posterior probability of correct alignment implemented in ProAlign, leaving 938 and 1474 nucleotide characters from 22 tRNA and 12S+16S rRNA genes, respectively. All genes were aligned separately and subsequently concatened into one dataset consisting of six partitions as suggested by PartitionFinder [
<xref ref-type="bibr" rid="B58">58</xref>
] (see Additional file
<xref ref-type="supplementary-material" rid="S1">1</xref>
); amino acids, 12S+16S rRNA genes and 22 concatenated tRNA genes (designated as 123
<sub>A</sub>
RT
<sub>n</sub>
). Missing nucleotides, e.g. tRNA-Thr and Pro genes for some species, were coded as missing characters. Levels of mutational saturation were calculated from p-distances for 1st, 2nd and 3rd codon positions using PAUP* Ver. 4.0b10 [
<xref ref-type="bibr" rid="B59">59</xref>
] (see Additional file
<xref ref-type="supplementary-material" rid="S2">2</xref>
). Since no mutational saturation was detected when including only myctophiforms, we constructed a second dataset consisting of only nucleotides from the 41 myctophiform species. This dataset included all mitogenomic nucleotides except the CR and ProAlign ambiguous alignment as already noted above (123
<sub>n</sub>
RT
<sub>n</sub>
).</p>
<p>Heuristic maximum likelihood phylogenetic analyses with 1000 non-parametric bootstrap replications for all nodes were performed in a single run with the sequential version of RAxML Ver. 7.2.8 [
<xref ref-type="bibr" rid="B60">60</xref>
,
<xref ref-type="bibr" rid="B61">61</xref>
] under the GTR+Γ+I substitution model [
<xref ref-type="bibr" rid="B62">62</xref>
] as chosen by Akaike and Bayesian information criteria [
<xref ref-type="bibr" rid="B63">63</xref>
,
<xref ref-type="bibr" rid="B64">64</xref>
] implemented in Modelgenerator [
<xref ref-type="bibr" rid="B65">65</xref>
]. A final ML optimization of every 5th bootstrapped tree for the highest scoring ML-tree is performed by RAxML under the specified GTR+Γ+I substitution model [
<xref ref-type="bibr" rid="B61">61</xref>
]. Partitions constructed from amino acids were analyzed with the mtREV model [
<xref ref-type="bibr" rid="B66">66</xref>
] chosen by Modelgenerator.</p>
<p>Bayesian analysis was performed on the datasets with MrBayes Ver. 3.2.1 [
<xref ref-type="bibr" rid="B67">67</xref>
] using the same partitions and models as described above. Maximum likelihood analyses were conducted on a Quad-core Mac Pro desktop computer whereas the Bayesian analyses were computed on the Bioportal computer cluster University of Oslo [
<xref ref-type="bibr" rid="B68">68</xref>
]. Convergence of chains and burn-in were determined using TRACER Ver. 1.5 [
<xref ref-type="bibr" rid="B69">69</xref>
].</p>
</sec>
<sec>
<title>Additional analyses and diaphini tRNA-met pseudogenes</title>
<p>All 41 myctophiform mitogenomes were scrutinized for mt features such as rearranged gene orders and INC-regions. All INC-sequences were analyzed for sequence similarity in relation to possible duplicated regions in the mitochondrial genome by using NCBI Blast [
<xref ref-type="bibr" rid="B54">54</xref>
], tRNAscan [
<xref ref-type="bibr" rid="B55">55</xref>
] and a series of decreasing assembly parameters in Sequencher [
<xref ref-type="bibr" rid="B53">53</xref>
]. Subsequently, alignments of INC-regions were examined for secondary folding patterns with LocARNA Ver. 1.5.2 [
<xref ref-type="bibr" rid="B70">70</xref>
] and RNAshapes Ver. 2.1.6 [
<xref ref-type="bibr" rid="B71">71</xref>
] in order to detect conserved regions. Secondary tRNA-structures were drawn with VARNA Ver. 3.8 [
<xref ref-type="bibr" rid="B72">72</xref>
]. In order to examine a putative associated gene order found in
<italic>Notolychnus</italic>
and species within the tribe Diaphini, neighbour-joining analyses were carried out including multiple myctophid tRNA-Met genes, putative duplicated diaphinid tRNA-Met genes (pseudogenes) and the two tRNA-Met genes observed in
<italic>Notolychnus</italic>
. Pairwise sequence variation of the pseudogenes and detected tRNA-Met genes in Diaphini were compared in order to validate the INC-regions as actual tRNA-Met pseudogenes, the latter expected to show higher substitution rates.</p>
</sec>
<sec>
<title>Testing alternative phylogenetic hypotheses</title>
<p>Topology comparison from morphological phylogenies and this study were tested by using the approximately unbiased log-likelihood test [
<xref ref-type="bibr" rid="B73">73</xref>
] implemented in CONSEL [
<xref ref-type="bibr" rid="B74">74</xref>
]. Constrained trees were produced with Mesquite [
<xref ref-type="bibr" rid="B75">75</xref>
] and per-site log-likelihood scores were produced using RAxML using the f –g option with best scoring ML-trees passed via –z option. Per-site log-likelihood scores and tree topologies were used as direct input into CONSEL outputting tree-specific ML-scores and associated
<italic>p</italic>
-values.</p>
</sec>
</sec>
<sec sec-type="results">
<title>Results</title>
<sec>
<title>Mitogenome organization</title>
<p>The organization of the mitogenome is unique in all fishes of the family Myctophidae, showing rearranged gene orders for either the whole family, tribal or subtribal clades, whereas Neoscopelidae show the canonical vertebrate gene order. All these features are summarized in Figure 
<xref ref-type="fig" rid="F3">3</xref>
with numbers 1–8 corresponding to rearranged gene orders (1–4) and INC-sequences (5–8).</p>
<p>1. WANYC-gene order found in all myctophids. The tRNA-Cys and Tyr have shifted positions and the putative O
<sub>L</sub>
-region was much longer than the usual vertebrate sequence in this region of about 30–40 base pairs (56–321 in myctophids included in this study). We were not able to find any conserved sequence blocks or secondary cloverleaf structure typical of tRNA genes [
<xref ref-type="bibr" rid="B76">76</xref>
] for this INC-region (see Additional file
<xref ref-type="supplementary-material" rid="S3">3</xref>
).</p>
<p>2. IMQM
<sub>ψ</sub>
gene order found in the tribe Diaphini. The tRNA-Gln and Met have shifted positions with the typical L- and H-strand coding intact (tRNA-Gln is coded on the L-strand and tRNA-Met coded on the H-strand). An INC-region (tRNA-Met pseudogene denoted as M
<sub>ψ</sub>
) is present between tRNA-Gln and ND1 in all diaphinid taxa included. A similar gene order was also observed for
<italic>Notolychnus valdiviae</italic>
although the INC-region is a tRNA-Met gene for this taxon (IMQM gene order). This particular gene rearrangement was analyzed and is discussed in detail below.</p>
<p>3. Relocation of tRNA-Glu resulting in gene order Cytb/T/E/P in a Lampanyctini subclade. Species included within
<italic>Lampanyctus</italic>
,
<italic>Nannobrachium</italic>
,
<italic>Stenobrachius</italic>
and
<italic>Triphoturus</italic>
all show tRNA-Glu pseudogenes in the canonical position between ND6 and Cytb, verified from sequence comparisons and partial secondary cloverleaf structures (see Additional file
<xref ref-type="supplementary-material" rid="S3">3</xref>
). We were only able to determine the position of the tRNA-Glu gene in two of the five taxa,
<italic>Nannobrachium ritteri</italic>
and
<italic>Lampanyctus crocodilus,</italic>
all showing the novel position of tRNA-Glu between tRNA-Thr and Pro. A highly variable region downstream the CR and multiple nucleotide repeats prevented us from determining this sequence in
<italic>Lampanyctus macdonaldi</italic>
,
<italic>Stenobrachius leucopsarus</italic>
and
<italic>Triphoturus nigrescens</italic>
.</p>
<p>4. Cytb/T/ND6/E/P gene order in a Myctophini subclade.
<italic>Myctophum punctatum</italic>
,
<italic>M. affine</italic>
and
<italic>M. nitidulum</italic>
showed this rearranged gene order whereas
<italic>M. asperum</italic>
and
<italic>M. orientale</italic>
showed the canonical gene order. Unknown technical issues prevented us from determining the sequence of ND6 in
<italic>M. punctatum</italic>
although the gene rearrangement is evident also in this species from ND5/Cytb contiguous sequences as opposed to the canonical ND5/ND6/E/Cytb gene order.</p>
<p>5. INC-sequence between Leu
<sup>UUR</sup>
and ND1 found in all myctophids. This spacer ranged from 45–79 base pairs and showed no secondary structure or sequence similarity to any other mt genes (see Additional file
<xref ref-type="supplementary-material" rid="S3">3</xref>
).</p>
<p>6. INC-sequence between ND1 and tRNA-Ile found in Electronini. This spacer showed significant length variation and we were unable to determine the whole sequence in
<italic>Protomyctophum arcticum</italic>
.</p>
<p>7. INC-sequence between ATP6 and CO3 found in all myctophids. This spacer was 288 base pairs long in
<italic>Notolychnus valdiviae</italic>
and ranged from 9–43 base pairs in all other myctophids. We found no mt gene similarity or secondary structure for this INC-region (see Additional file
<xref ref-type="supplementary-material" rid="S3">3</xref>
).</p>
<p>8. INC-sequence associated with tRNA-Gly found in a Myctophini subclade. This INC-region is treated as a character state for the clade comprising
<italic>Benthosema</italic>
and
<italic>Diogenichthys</italic>
although the exact position varies between the two genera. The three species of
<italic>Benthosema</italic>
included showed the INC-region located upstream the tRNA-Gly whereas in
<italic>Diogenichthys atlanticus</italic>
, the spacer was located downstream tRNA-Gly.
<italic>Electrona antarctica</italic>
showed a different organization with two detected tRNA-Gly genes separated by a large INC-region. We found no sequence similarity to other mt genes for this region or any secondary folding structures (see Additional file
<xref ref-type="supplementary-material" rid="S3">3</xref>
).</p>
<fig id="F3" position="float">
<label>Figure 3</label>
<caption>
<p>
<bold>Typical gene orders in vertebrates and Neoscopelidae with eight unique gene orders and INC-regions found in Myctophidae.</bold>
1
<bold></bold>
4 show rearranged gene orders and 5–8 show INC-sequences with all eight features treated as clade-defining synapomorphies. Taxon-specific gene rearrangements are shown at the bottom. All INC-regions are shown in grey with number of base pairs noted if known. Mt information depicted is provided from all included taxa within the clades except for the rearrangement in 3 (information from
<italic>N. ritteri</italic>
and
<italic>L. crocodilus</italic>
), in 4 (information from
<italic>M. affine</italic>
and
<italic>M. nitidulum</italic>
) and in 6 (information from
<italic>K. anderssoni</italic>
and
<italic>E. antarctica</italic>
). The missing information for some included taxa is due to unsuccessful amplification of particular regions. All eight features are discussed in the text. Abbreviations follow common mt usage and colorcoding is signifying the same clades and defining gene orders throughout all figures in this article.</p>
</caption>
<graphic xlink:href="1471-2148-13-111-3"></graphic>
</fig>
<p>Additional gene orders and INC-sequences were observed for various myctophid species although none of the rearrangements were distributed across multiple taxa at this point. These additional gene order rearrangements and INC-sequences are also presented in Figure 
<xref ref-type="fig" rid="F3">3</xref>
. Detailed information for rearranged gene orders such as length of INC-sequences and missing regions is presented in the supplementary material (see Additional file
<xref ref-type="supplementary-material" rid="S4">4</xref>
). The highly polymorphic control region (CR) was consistently problematic to amplify or sequence, resulting in parts of the CR missing for most species as well as flanking tRNA-Thr and -Pro for some species.</p>
</sec>
<sec>
<title>Mitogenome sequences</title>
<p>ML- and Bayesian analyses of the mitogenomic dataset consisting of 87 taxa resolved a phylogenetic tree of eu- and neoteleostean orders as presented in Figure 
<xref ref-type="fig" rid="F4">4</xref>
. Myctophiformes was resolved as the sister clade to Lampriformes supported by a BS-value of only 68% whereas the BPP-value was 93%. Both analyses conducted for this study showed this relationship; however, considering the long basal branches of Lampriformes and Myctophiformes, we conclude that the phylogenetic position of Myctophiformes, from mitogenomic evidence, in relation to basal neoteleostean orders and Acanthomorpha remains ambiguous. This is supported by comparison to previous mitogenomic studies [
<xref ref-type="bibr" rid="B33">33</xref>
,
<xref ref-type="bibr" rid="B43">43</xref>
,
<xref ref-type="bibr" rid="B46">46</xref>
,
<xref ref-type="bibr" rid="B77">77</xref>
] and nuclear genes [
<xref ref-type="bibr" rid="B78">78</xref>
]. Although we have included all neoteleostean orders in this study (Figure 
<xref ref-type="fig" rid="F4">4</xref>
), this issue requires the inclusion of additional mitogenomes from all neoteleostean orders, and analyses carried out using newly developed models accounting for site-specific modulations of the amino acid replacement process, such as for example the CAT mixture model [
<xref ref-type="bibr" rid="B79">79</xref>
] implemented in phylobayes [
<xref ref-type="bibr" rid="B80">80</xref>
]. However, the latter program does not implement mixture of characters preventing the use of all information included in the mt genome, making this analysis premature and is therefore not employed in this study.</p>
<fig id="F4" position="float">
<label>Figure 4</label>
<caption>
<p>
<bold>Mitogenomic Neoteleostei phylogeny (123</bold>
<sub>
<bold>A</bold>
</sub>
<bold>RT</bold>
<sub>
<bold>n</bold>
</sub>
<bold>) rooted with Euteleostei.</bold>
Branch support from 1000 Bootstrap replicates and Bayesian posterior probabilities noted only if values are less than 100 – separated by a slash (
<bold>/</bold>
), respectively. Three topological differences between the Bayesian- and ML-analyses are noted with bootstrap values included. Ten distinct myctophid lineages are noted (A-J) with respect to current tribal classification.</p>
</caption>
<graphic xlink:href="1471-2148-13-111-4"></graphic>
</fig>
<p>The myctophiform ingroup topology from the two datasets analyzed (87 taxa: 123
<sub>A</sub>
RT
<sub>n</sub>
and 41 taxa: 123
<sub>n</sub>
RT
<sub>n</sub>
) showed differences related to lampanyctine tribal relationships (Figures 
<xref ref-type="fig" rid="F4">4</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
). ML- and Bayesian analyses of the 87 taxa also showed incongruence concerning the phylogenetic position of
<italic>Triphoturus nigrescens</italic>
(Figure 
<xref ref-type="fig" rid="F4">4</xref>
).</p>
<fig id="F5" position="float">
<label>Figure 5</label>
<caption>
<p>
<bold>Mitogenomic Myctophiformes phylogeny (123</bold>
<sub>
<bold>n</bold>
</sub>
<bold>RT</bold>
<sub>
<bold>n</bold>
</sub>
<bold>) rooted with Neoscopelidae.</bold>
Branch support from 1000 Bootstrap replicates and Bayesian posterior probabilities noted only if values are less than 100 – separated by a slash (/), respectively. One topological difference between the Bayesian- and ML-analyses is noted with bootstrap value included. Ten distinct myctophid lineages are noted (A-J) with respect to current tribal classification.</p>
</caption>
<graphic xlink:href="1471-2148-13-111-5"></graphic>
</fig>
<p>Neoscopelidae and Myctophidae were both recovered as monophyletic with
<italic>Scopenlengys tristis</italic>
recovered as the sister taxon to the remaining neoscopelids (low support) and
<italic>Notolychnus valdiviae</italic>
was recovered as the sister taxon to the remaining myctophids (high support). Myctophine and lampanyctine subfamilies were also recovered as monophyletic in all analyses. The tribe Diaphini was recovered as the sister lineage to the other lampanyctines, found in all analyses although with low support values, despite the unresolved tribal relationships within the remaining lampanyctines. The tribe Electronini was recovered as the sister lineage to the other myctophines with maximum levels of support and a clade consisting of
<italic>Benthosema</italic>
spp. and
<italic>Diogenichthys atlanticus</italic>
was resolved as the sister lineage to the tribes Gonichthyini and Myctophini. Ten myctophid lineages are recognized from this study and are noted according to current tribal classification as A-J in Figures 
<xref ref-type="fig" rid="F4">4</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
. We note that deep myctophiform- and tribe-defining nodes were consistently found in all analyses, although support values indicate uncertainty concerning basal neoscopelid-, basal lampanyctine- and to a lesser degree basal myctophine phylogenetic relationships. Tribal relationships within Lampanyctinae (clades D, E and F) proved to be the most problematic part of myctophid phylogenetics from mitogenomics, showing incongruent results between datasets employed and mode of analysis (Figures 
<xref ref-type="fig" rid="F4">4</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
).</p>
</sec>
<sec>
<title>Diaphini tRNA-Met pseudogenes</title>
<p>All taxa within the tribe Diaphini show an IMQ-gene order in addition to an INC-region upstream from ND2 ranging from 62 to 68 base pairs. The INC-region shows varying degrees of sequence similarity to detected tRNA-Met genes with most tRNA secondary structures missing only the D- and variable loops of the typical tRNA gene cloverleaf secondary structure (Figure 
<xref ref-type="fig" rid="F6">6</xref>
). Pairwise distances for tRNA-Met pseudogenes (INC-regions) were all much higher than for the detected tRNA-Met genes (Figure 
<xref ref-type="fig" rid="F7">7</xref>
). In view of the IMQM
<sub>ψ</sub>
gene order and putative tRNA-Met duplication in Diaphini, an interesting finding is observed in the monotypic basal branching taxon
<italic>Notolychnus valdiviae,</italic>
exhibiting two identified tRNA-Met genes resulting in an IMQM gene order. The two tRNA-Met genes both show the CAU-anticodon although they are highly divergent with respect to sequence similarity (~20% p-distance). Neigbour-joining distance method analyses of the tRNA-Met genes and their pseudogene counterparts showed clear phylogenetic structure in the diaphinid pseudogenes (Figure 
<xref ref-type="fig" rid="F6">6</xref>
). Including the two tRNA-Met genes observed in
<italic>Notolychnus</italic>
(IMQM gene order) in the analyses, in order to examine a possible connection to Diaphini (IMQM
<sub>ψ</sub>
gene order) concerning this gene-order rearrangement, showed no phylogenetic relationship between these taxa (Figure 
<xref ref-type="fig" rid="F6">6</xref>
).</p>
<fig id="F6" position="float">
<label>Figure 6</label>
<caption>
<p>
<bold>K2P-distance analyses of tRNA-Met genes and the tRNA-Met pseudogenes found in Diaphini and </bold>
<bold>
<italic>Notolychnus. </italic>
</bold>
<bold>A</bold>
. Analysis done with randomly selected myctophid tRNA-Met genes using a neoscopelid outgroup.
<bold>B</bold>
. Analysis done with only diaphinid taxa and
<italic>Notolychnus</italic>
using a neoscopelid outgroup. Note the better phylogenetic structure in the pseudogenes compared to their counterpart tRNA-Met genes.
<bold>C</bold>
. Mitogenomic cladogram showing the IMQM
<sub>ψ</sub>
– region in Diaphini. The truncated branch of
<italic>Notolychnus</italic>
signifies a phylogenetic position not corroborated from DNA sequences or tRNA-Met/tRNA-Met pseudogene distance analyses. Shaded areas represent INC-regions with number of base pairs noted for each taxon. Note the “gradual” removal of the INC-regions in diaphinid taxa except the tRNA-Met pseudogene (dark shaded). Secondary folding structures are presented for
<italic>Notolychnus</italic>
tRNA-Met1 and Met2 and all diaphinid tRNA-Met pseudogenes.</p>
</caption>
<graphic xlink:href="1471-2148-13-111-6"></graphic>
</fig>
<fig id="F7" position="float">
<label>Figure 7</label>
<caption>
<p>
<bold>Fifteen pairwise sequence divergences of tRNA-Met and tRNA-Met pseudogenes of the six diaphinid taxa included in this study.</bold>
Note the statistical significant higher sequence divergences in the tRNA-Met pseudogenes compared to the tRNA-met genes (
<italic>t</italic>
-test, df=13, P < 0.05).</p>
</caption>
<graphic xlink:href="1471-2148-13-111-7"></graphic>
</fig>
</sec>
<sec>
<title>Testing alternative phylogenetic hypotheses</title>
<p>Comparing constrained topologies according to morphology with the best scoring ML-tree from this study rejected a Notolychnini-Lampanyctinae relationship (
<italic>P</italic>
= 0.024; Table 
<xref ref-type="table" rid="T2">2</xref>
) as found from most morphological studies (Figure 
<xref ref-type="fig" rid="F2">2</xref>
). A Notolychnini-Myctophinae relationship could not be rejected (
<italic>P</italic>
= 0.151) and the topology with Notolychnini constrained within Diaphini as indicated from the tRNA-Met duplication event was clearly not rejected (
<italic>P</italic>
= 0.407).</p>
<table-wrap position="float" id="T2">
<label>Table 2</label>
<caption>
<p>Statistical comparisons between phylogenetic hypotheses using the AU-test (41 taxa Myctophiformes dataset)</p>
</caption>
<table frame="hsides" rules="groups" border="1">
<colgroup>
<col align="left"></col>
<col align="left"></col>
<col align="left"></col>
<col align="left"></col>
</colgroup>
<thead valign="top">
<tr>
<th align="left">
<bold>Topology</bold>
</th>
<th align="left">
<bold>-ln L</bold>
</th>
<th align="left">
<bold>Δ ln L</bold>
</th>
<th align="left">
<bold>AU*</bold>
</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="left" valign="bottom">(Notolychnus (Lampanyctinae, Myctophinae))
<hr></hr>
</td>
<td align="left" valign="bottom">221112.283340
<hr></hr>
</td>
<td align="left" valign="bottom">Best
<hr></hr>
</td>
<td align="left" valign="bottom">0.733
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">((Notolychnus, Lampanyctinae) Myctophinae))
<hr></hr>
</td>
<td align="left" valign="bottom">221130.952336
<hr></hr>
</td>
<td align="left" valign="bottom">18.668996
<hr></hr>
</td>
<td align="left" valign="bottom">0.024*
<hr></hr>
</td>
</tr>
<tr>
<td align="left" valign="bottom">(Notolychnus, Myctophinae) Lampanyctinae)
<hr></hr>
</td>
<td align="left" valign="bottom">221125.980254
<hr></hr>
</td>
<td align="left" valign="bottom">13.696914
<hr></hr>
</td>
<td align="left" valign="bottom">0.151
<hr></hr>
</td>
</tr>
<tr>
<td align="left">(((Notolychnus, Diaphini) Lampanyctinae) Myctophinae)</td>
<td align="left">221118.943061</td>
<td align="left">06.659721</td>
<td align="left">0.407</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<p>*
<italic>P</italic>
< 0.05 = constrained topology rejected at the 5% confidence level.</p>
</table-wrap-foot>
</table-wrap>
</sec>
</sec>
<sec sec-type="discussion">
<title>Discussion</title>
<sec>
<title>Monophyly of myctophiformes and the two families</title>
<p>This study recovered a monophyletic Myctophiformes and monophyletic families with Neoscopelidae as the sister family of the highly diversified Myctophidae (Figures 
<xref ref-type="fig" rid="F4">4</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
; [
<xref ref-type="bibr" rid="B3">3</xref>
,
<xref ref-type="bibr" rid="B5">5</xref>
,
<xref ref-type="bibr" rid="B81">81</xref>
,
<xref ref-type="bibr" rid="B82">82</xref>
] although see Rosen [
<xref ref-type="bibr" rid="B83">83</xref>
]). We found
<italic>Scopelengys tristis</italic>
was a sister taxon to the remaining neoscopelids, including the monotypic endemic Sulu Sea taxon
<italic>Solivomer arenidens</italic>
[
<xref ref-type="bibr" rid="B84">84</xref>
], in congruence with morphology (Figure 
<xref ref-type="fig" rid="F2">2</xref>
). Stiassny [
<xref ref-type="bibr" rid="B5">5</xref>
] presented characters in support for
<italic>Scopelengys</italic>
as sister taxa to the remaining neoscopelids, although she did note a possibility of apparently derived characters in
<italic>Neoscopelus</italic>
and
<italic>Solivomer</italic>
being secondarily reduced in
<italic>Scopelengys</italic>
. Analyses from the present study are congruent on this matter although support values are alarmingly low concerning the phylogenetic position of
<italic>Solivomer arenidens</italic>
. We found
<italic>Notolychnus valdiviae</italic>
to be the sister taxon to the remaining myctophids; however, morphological character states of
<italic>Notolychnus</italic>
such as dorsally located photophores, small larvae without pigmentation and a reduced otolith, have been extremely difficult to interpret concerning early myctophid divergence. Photophores are present in all myctophids except
<italic>Taaningichthys paurolychnus</italic>
and present only in one Neoscopelidae genus
<italic>Neoscopelus.</italic>
Our results show both of these genera nested within their respective families and the evolution of photophores within Myctophiformes is difficult to assess, i.e. whether photophores have evolved once or twice within ancestral myctophiforms. This question is pertinent from morphological considerations on photophore structure in Myctophidae and Neoscopelidae, as it is quite different in the two groups [
<xref ref-type="bibr" rid="B85">85</xref>
,
<xref ref-type="bibr" rid="B86">86</xref>
]. The phylogenetic positions of
<italic>Scopelengys</italic>
/
<italic>Solivomer</italic>
and
<italic>Notolychnus</italic>
in this study revives an interesting observation by Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] concerning parallel evolutionary trends for
<italic>Solivomer/</italic>
Lampanyctinae and for
<italic>Neoscopelus</italic>
/Myctophinae, regarding changes in jaw length and characters possibly associated with these changes. These characters have been difficult to interpret in terms of polarity and this study supports long jaw length as the plesiomorphic state in Neoscopelidae and Myctophidae from the basal branchings of
<italic>Scopelengys</italic>
/
<italic>Solivomer</italic>
and
<italic>Notolychnus</italic>
. Also, Stiassny [
<xref ref-type="bibr" rid="B5">5</xref>
] noted that important myctophiform character states like elongated jaw length and raised photophores show ambiguity concerning early myctophiform divergence.</p>
<p>A monophyletic Myctophidae was strongly supported by mitogenome sequences, a WANYC gene order and an INC-spacer between tRNA-Leu
<sup>UUR</sup>
and ND1 (Figures 
<xref ref-type="fig" rid="F3">3</xref>
and
<xref ref-type="fig" rid="F4">4</xref>
). Rearrangement of the canonical vertebrate WANCY gene order was shared by all myctophids with the tRNA-Cys relocated downstream resulting in a synapomorphic WANYC gene order (Figure 
<xref ref-type="fig" rid="F5">5</xref>
). A prolonged sequence (putative O
<sub>L</sub>
+ INC-sequence) in the canonical position of the putative origin of L-strand replication was found within all myctophids (see Additional file
<xref ref-type="supplementary-material" rid="S4">4</xref>
), although fishes within the tribe Electronini were difficult to determine due to additionally rearranged gene orders in this region (Figure 
<xref ref-type="fig" rid="F3">3</xref>
). An INC-region between the tRNA-Leu
<sup>UUR</sup>
and ND1 also represents synapomorphic evidence for Myctophidae (Figure 
<xref ref-type="fig" rid="F3">3</xref>
). The size of this spacer ranged from 45–79 base pairs and we found no obvious tendency regarding size change for this INC-region (see Additional file
<xref ref-type="supplementary-material" rid="S4">4</xref>
). Neither the O
<sub>L</sub>
+ INC-region nor the Leu
<sup>UUR</sup>
/ND1 spacers showed sequence similarity in any species to any other mitochondrial genes. Neither could they be folded into clover shape secondary structures, so their origins are elusive. On the other hand, assembly of the INC-spacers showed clear sequence similarity within genera indicating the INC-regions to be inherited synapomorphic characters within the family. Smith et al. [
<xref ref-type="bibr" rid="B87">87</xref>
] reported a length expansion for a similar small INC-sequence located between tRNA-Val and 12S rRNA in a clade of serranid fairy basslet fishes (Serranidae). They showed the INC-sequence to be homologous for all genera from simultaneous analysis of nucleotides and gene order – a very difficult task for myctophids considering multiple gene order rearrangements and putative associated INC-sequences. The third myctophid synapomorphy from this study is an INC-region between ATP6 and CO3 ranging from 288 base pairs in
<italic>Notolychnus</italic>
to 9–43 base pairs in the remaining myctophids included (see Additional file
<xref ref-type="supplementary-material" rid="S4">4</xref>
). No conserved sequence motifs were found in this region. This is a peculiar spacer in the sense that the canonical vertebrate gene order for this region is ATP6/CO3 with no tRNA gene acting as punctuation mark for RNA processing.</p>
</sec>
<sec>
<title>Monophyly of the two subfamilies and five tribes</title>
<p>The monophyly of the two subfamilies Lampanyctinae and Myctophinae was recovered from all analyses, although the low support values associated with this split are incomprehensible from morphological characters unambigously delimiting these two groups. As with the Myctophidae, the two subfamilies are fully supported by adult and larval morphology and no discussion is needed – character states are presented by Moser & Ahlstrom [
<xref ref-type="bibr" rid="B28">28</xref>
], Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] and Stiassny [
<xref ref-type="bibr" rid="B5">5</xref>
]. A well supported result from this study, differing from morphology, is the phylogenetic position of the tribe Diaphini within Lampanyctinae, resolved as sister tribe to the other lampanyctines. Adult and larval morphology within Lampanyctinae show incongruences and both hypotheses are different compared to our molecular results (Figures 
<xref ref-type="fig" rid="F2">2</xref>
,
<xref ref-type="fig" rid="F4">4</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
).This shows problematic morphological characters, lampanyctine larval characters are noted as few and tentative in previous larval studies [
<xref ref-type="bibr" rid="B30">30</xref>
,
<xref ref-type="bibr" rid="B88">88</xref>
] and of little use above genus level, as well as molecular characters, no unique gene orders connect tribes and the mitogenomic phylogenetic sequence information is insufficient, as witnessed from the three different resolved Lampanyctinae topologies from this study (Figures 
<xref ref-type="fig" rid="F4">4</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
).</p>
<p>Monophyly of the tribes Notolychnini (clade A in Figures 
<xref ref-type="fig" rid="F4">4</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
), Diaphini (B), Gymnoscopelini (C), Lampanyctini (D+E+F), Electronini (G) and two subclades of Myctophini (H and I+J) are strongly supported from all mitogenomic DNA sequence analyses and various mt gene orders discussed below. Only the node delimiting the tribe Diaphini (B) show relatively low support values. The tribe Gonichthyini is tentatively placed from a single taxon as nested within a Myctophini subclade consisting of ctenoid-scaled species. Mitogenomic DNA sequence data from this study supports recognition of 10 mutually exclusive clades within Myctophidae (Figures 
<xref ref-type="fig" rid="F4">4</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
).</p>
</sec>
<sec>
<title>Phylogeny of tribes and genera</title>
<p>Students of lanternfish phylogenetics will note the position of
<italic>Notolychnus</italic>
(clade A) resolved as sister taxon to the remaining family Myctophidae from mitogenome sequences. Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] tentatively placed
<italic>Notolychnus</italic>
as sister taxon to the subfamily Lampanyctinae (Figure 
<xref ref-type="fig" rid="F2">2</xref>
A). Subsequent parsimony analysis of the same data by Paxton et al. [
<xref ref-type="bibr" rid="B30">30</xref>
], including additional larval characters [
<xref ref-type="bibr" rid="B27">27</xref>
-
<xref ref-type="bibr" rid="B29">29</xref>
], placed
<italic>Notolychnus</italic>
in an unresolved Myctophinae-Notolychnini-Lampanyctinae trifurcation (Figure 
<xref ref-type="fig" rid="F2">2</xref>
D). Subsequently, Stiassny [
<xref ref-type="bibr" rid="B5">5</xref>
] added four additional characters and reanalyzed the aforementioned data with a similar result, showing
<italic>Notolychnus</italic>
as the sister taxon to the remaining lampanyctines (Figure 
<xref ref-type="fig" rid="F2">2</xref>
E). Most recently, Yamaguchi [
<xref ref-type="bibr" rid="B24">24</xref>
] scrutinized previously published characters (Figure 
<xref ref-type="fig" rid="F2">2</xref>
A-E) once again and reanalyzed the data, resulting in a largely unresolved myctophid tree (Figure 
<xref ref-type="fig" rid="F2">2</xref>
F). However, the position of
<italic>Notolychnus</italic>
was congruent with Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] and Stiassny [
<xref ref-type="bibr" rid="B5">5</xref>
]. The novel phylogenetic position of
<italic>Notolychnus</italic>
from this study, as the basal branching in Myctophidae, highlight the past issues concerning subfamilial affiliation and partly explains the lack of phylogenetically informative morphological characters in relation to the two subfamilies.</p>
<p>A monophyletic Diaphini (clade B) was well supported from both sequences and a unique IMQ-gene order (Figure 
<xref ref-type="fig" rid="F3">3</xref>
), all in complete agreement with morphology, showing
<italic>Lobianchia</italic>
(2 species) as sister group of the very speciose
<italic>Diaphus</italic>
(78 species) [
<xref ref-type="bibr" rid="B89">89</xref>
-
<xref ref-type="bibr" rid="B91">91</xref>
]. Four species-groups of
<italic>Diaphus</italic>
have been suggested from the presence of Dn-Vn, Ant, Suo and So photophores (for photophore terminology, see Figure 
<xref ref-type="fig" rid="F1">1</xref>
) [
<xref ref-type="bibr" rid="B91">91</xref>
]; however, our five species of
<italic>Diaphus</italic>
included were insufficient to validate anything on this matter. One very rare monotypic taxon
<italic>Idiolychnus urolampus</italic>
, missing from our study, was transferred from
<italic>Diaphus</italic>
to
<italic>Lobianchia</italic>
by Bolin [
<xref ref-type="bibr" rid="B92">92</xref>
] and most lately erected into its own genus
<italic>Idiolychnus</italic>
by the position of VO
<sub>3</sub>
and possession of two SAOs [
<xref ref-type="bibr" rid="B93">93</xref>
].</p>
<p>Gymnoscopelini (clade C) sensu Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] was found to be monophyletic although not fully congruent with Ahlstrom et al. [
<xref ref-type="bibr" rid="B88">88</xref>
]. Unfortunately, the monotypic taxon
<italic>Lampanyctodes hectoris</italic>
is not included in this study, a myctophid showing ambiguous morphological characters concerning tribal placement. Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] noted
<italic>Lampanyctodes</italic>
as a possible early divergence based on several characters shared with the most plesiomorphic diaphinid genus
<italic>Lobianchia</italic>
. Conversely, Ahlstrom et al. [
<xref ref-type="bibr" rid="B88">88</xref>
] noted
<italic>Lampanyctodes</italic>
as being specialized within Gymnoscopelini from a similar larval form and development to that found within
<italic>Lampadena</italic>
, arguing that characters reflect habitat instead of true phylogeny.</p>
<p>
<italic>Ceratoscopelus</italic>
and
<italic>Bolinichthys</italic>
formed a clade (D) as either sister group to the remaining lampanyctines or included within them (Figures 
<xref ref-type="fig" rid="F4">4</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
, respectively). Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] included these genera within Lampanyctini as closely related, whereas Ahlstrom et al. [
<xref ref-type="bibr" rid="B88">88</xref>
] placed them within Gymnoscopelini. Our study supports inclusion in Lampanyctini despite unresolved intrarelationships of this tribe. The clade itself is very distinct in terms of mt sequences and supported by both osteology [
<xref ref-type="bibr" rid="B3">3</xref>
] and larval development [
<xref ref-type="bibr" rid="B30">30</xref>
].</p>
<p>A tRNA-Glu gene could be detected in the canonical vertebrate position between ND6 and Cytb in all myctophids included in this study, except in a Lampanyctini subclade (E) comprising
<italic>Stenobrachius</italic>
,
<italic>Triphoturus</italic>
,
<italic>Nannobrachium</italic>
and
<italic>Lampanyctus</italic>
(Figures 
<xref ref-type="fig" rid="F3">3</xref>
). In
<italic>Nannobrachium ritteri</italic>
and
<italic>Lampanyctus crocodilus,</italic>
we were able to determine a novel location of tRNA-Glu in the highly polymorphic region downstream Cytb including the tRNA-Thr and Pro genes, the CR and various INC-regions. Sequence similarities between tRNA-Glu from
<italic>N. ritteri</italic>
and
<italic>L. crocodilus</italic>
and the INC-regions between ND6 and Cytb were evident from sequence comparisons and putative secondary structures (see Additional file
<xref ref-type="supplementary-material" rid="S3">3</xref>
). Interestingly, this INC-region showed no sign of redundancy since all five taxa have retained a fragment equal in size to the typical length of tRNA genes (68–73 base pairs). We tentatively assign this gene order to the lampanyctine subclade E and note that this clade is congruent with Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] and Paxton et al. [
<xref ref-type="bibr" rid="B30">30</xref>
], although the latter notes that no synapomorphic character is present to actually define the clade. We expect this feature to be found within the unsampled genus
<italic>Parvilux</italic>
[
<xref ref-type="bibr" rid="B94">94</xref>
] as well. A new position of tRNA-Glu and retention of an INC-sequence between ND6 and Cytb has also been found in other vertebrates, e.g. the amphisbaenian reptile family Bipedidae [
<xref ref-type="bibr" rid="B95">95</xref>
], and seems to be one of the most common gene order rearrangements in vertebrates.</p>
<p>A clade consisting of
<italic>Lampadena</italic>
and
<italic>Taaningichthys</italic>
(F) is found monophyletic and completely congruent with morphology
<italic>. Taaningichthys</italic>
was previously a part of the genus
<italic>Lampadena</italic>
[
<xref ref-type="bibr" rid="B4">4</xref>
] although it was separated by Bolin [
<xref ref-type="bibr" rid="B92">92</xref>
] based on characters such as reduced number of photophores and flaccid body structure. Nafpaktitis and Paxton [
<xref ref-type="bibr" rid="B96">96</xref>
] noted that
<italic>Lampadena chavesi</italic>
,
<italic>L. dea</italic>
and
<italic>L. speculigera</italic>
were more closely related to
<italic>Taaningichthys</italic>
than the remaining species of
<italic>Lampadena</italic>
based on expanded neural arches on the anterior vertebrae as found in
<italic>Taaningichthys</italic>
. In fact, Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] noted that discriminating characters between the two genera were hard to find.
<italic>Lampadena</italic>
and especially
<italic>Taaningichthys</italic>
are among the largest and deepest living myctophids, with the latter showing reduction of the lateral line and in the number of body photophores. This apparent reductional trend has resulted in one taxon,
<italic>Taaningichthys paurolychnus</italic>
, having lost all body photophores although retained the supra- and infracaudal glands.
<italic>Lampadena yaquinae</italic>
is found nested within the
<italic>Lampadena</italic>
species included and corroborates Paxton [
<xref ref-type="bibr" rid="B97">97</xref>
] in synonymizing
<italic>Dorsadena</italic>
[
<xref ref-type="bibr" rid="B98">98</xref>
] with
<italic>Lampadena</italic>
. Likewise, the subgenera
<italic>Lampadena</italic>
and
<italic>Lychnophora</italic>
proposed by Fraser-Brunner [
<xref ref-type="bibr" rid="B4">4</xref>
], supported by only an elevated PO
<sub>4</sub>
[
<xref ref-type="bibr" rid="B96">96</xref>
], are not supported from this study. Increased mitogenomic taxon sampling of these two genera is interesting concerning evolution of photophores within these deep dwelling genera showing atypical character states within the Myctophidae [
<xref ref-type="bibr" rid="B99">99</xref>
].</p>
<p>The tribe Electronini (G), erected from low-leveled PVO and PLO photophores [
<xref ref-type="bibr" rid="B25">25</xref>
], composes a subset of the tribe Myctophini as recognized by Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
]. Our results clearly support Electronini as a separate tribe [
<xref ref-type="bibr" rid="B100">100</xref>
] from both DNA sequences and long (>200 base pairs) synapomorphic INC-regions upstream the tRNA-Ile gene (Figures 
<xref ref-type="fig" rid="F3">3</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
). We recovered a sister relationship of Electronini to the rest of Myctophinae, a result also presented by Paxton et al. [
<xref ref-type="bibr" rid="B30">30</xref>
], although cladistic analyses of morphological characters failed to recover this result (Figures 
<xref ref-type="fig" rid="F2">2</xref>
E-F).</p>
<p>The IQM-regions of species included in the genera
<italic>Benthosema</italic>
and
<italic>Diogenichthys</italic>
(clade H) were peculiar and the IQM-region and WANYC-region of both Electronini and
<italic>Benthosema-Diogenichthys</italic>
were highly differentiated compared to all other myctophids.
<italic>Benthosema glaciale</italic>
showed new positions of tRNA-Gln and -Met to the downstream region of the WANYC-region (Figure 
<xref ref-type="fig" rid="F3">3</xref>
) representing a novel gene order in vertebrate mitogenomes.
<italic>Diogenichthys atlanticus</italic>
showed an IQMM gene order and the canonical vertebrate gene order was observed within
<italic>Benthosema pterotum</italic>
and
<italic>B. fibulatum</italic>
. Taxon sampling prevents us from discussing rearrangements for these taxa at this point; however, a close relationship between the two genera is consistent with morphology.
<italic>Diogenichthys</italic>
was separated from
<italic>Benthosema</italic>
by ventrally leveled Prc 1 and 2 in addition to various jaw differences and hooked dentary teeth, a character state only found within lampanyctines. Similar to
<italic>Notolychnus, D. atlanticus</italic>
is a diminutive species with adult maximum size < 30 millimeters and could represent a problematic branch within this clade considering character states related to miniaturization. Diminutive species are also found within the genus
<italic>Diaphus</italic>
. The
<italic>Benthosema</italic>
-
<italic>Diogenichthys</italic>
clade was supported from DNA sequences and an INC-region up- or downstream tRNA-Gly; all three species of
<italic>Benthosema</italic>
showed an INC-region flanked by an upstream CO3 gene and downstream tRNA-Gly. In addition,
<italic>B. glaciale</italic>
showed an INC-region after tRNA-Gly. In
<italic>D. atlanticus</italic>
, however, the INC-spacer was located downstream from the tRNA-Gly gene (Figure 
<xref ref-type="fig" rid="F3">3</xref>
). Interestingly, two tRNA-Gly genes separated by a large INC-region were observed in this same region in
<italic>Electrona antarctica</italic>
, although none of the INC-regions could be shown to have any sequence similarity to each other or to tRNA-Gly. We have assumed an INC-sequence associated with tRNA-Gly as a character state for the
<italic>Benthosema-Diogenichthys</italic>
clade, although with reservations. In view of
<italic>E. antarctica</italic>
possesing two tRNA-Gly genes we note that an association of this region in
<italic>E. antarctica</italic>
,
<italic>D. atlanticus</italic>
and
<italic>Benthosema</italic>
spp. is plausible and should be elucidated with increased mitogenomic taxon sampling.</p>
<p>The tribe Myctophini was not supported from this study as the sister clade to Electronini, because the single representative of the tribe Gonichthyini (
<italic>Centrobranchus choerocephalus</italic>
) included in this study renders Myctophini paraphyletic. We note that broader taxonomic representation of the genera
<italic>Gonichthys, Centrobranchus, Tarletonbeania</italic>
and
<italic>Loweina</italic>
is necessary concerning phylogenetic position of Gonichthyini within Myctophinae. However, evidence from mt sequences and a unique gene order in part of Myctophini (Figure 
<xref ref-type="fig" rid="F5">5</xref>
) is consistent with the results from Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] stating that”..
<italic>A number of characters suggest that the</italic>
Myctophum-Symbolophorus
<italic>line gave rise to the slendertails</italic>
[ed. tribe Gonichthyini]..”. Additional evidence for this result comes from the non-monophyly of Gonichthyini based on larval characters [
<xref ref-type="bibr" rid="B27">27</xref>
] (Figure 
<xref ref-type="fig" rid="F2">2</xref>
C) and the analyses by Stiassny [
<xref ref-type="bibr" rid="B5">5</xref>
] and Yamaguchi [
<xref ref-type="bibr" rid="B24">24</xref>
] (Figure 
<xref ref-type="fig" rid="F2">2</xref>
E-F) showing
<italic>Symbolophorus</italic>
a basal branching within Gonichthyini. This study confidently resolves the genus
<italic>Myctophum</italic>
into two different clades from mitogenome sequences and also from a unique gene order involving ND6, tRNA-Glu and Cytb (Figures 
<xref ref-type="fig" rid="F3">3</xref>
,
<xref ref-type="fig" rid="F5">5</xref>
).
<italic>Myctophum affine</italic>
,
<italic>M. punctatum</italic>
and
<italic>M. nitidulum</italic>
(clade I) showed this particular gene arrangement whereas
<italic>M. asperum</italic>
and
<italic>M. orientale</italic>
showed the typical myctophid gene order also found in
<italic>Centrobranchus choerocephalus</italic>
and
<italic>Symbolophorus californiensis</italic>
, the latter four forming a well supported clade J (Figures 
<xref ref-type="fig" rid="F4">4</xref>
and
<xref ref-type="fig" rid="F5">5</xref>
). Two species-groups are currently recognized in the genus
<italic>Myctophum</italic>
based on cycloid versus ctenoid scales [
<xref ref-type="bibr" rid="B101">101</xref>
] corresponding to the two separated groups of
<italic>Myctophum</italic>
found in this study. Moser and Ahlstrom [
<xref ref-type="bibr" rid="B27">27</xref>
] discussed eye shape within
<italic>Myctophum</italic>
and noted this genus as difficult to distinguish phylogenetically, with stalked eyes usually absent, although present in e.g.
<italic>Myctophum nitidulum</italic>
and
<italic>M. punctatum</italic>
corresponding to the cycloid-scaled
<italic>Myctophum</italic>
lineage from this study. This result is, however, complicated by stalked eyes also being found in all species of
<italic>Symbolophorus</italic>
[
<xref ref-type="bibr" rid="B27">27</xref>
]. Clearly, mitogenome sequences and a unique gene order in the cycloid-scaled species of
<italic>Myctophum</italic>
strongly suggest that the two groups are in fact different lineages within Myctophini and that the tribe Gonichthyini is nested within the ctenoid-scaled group. This indicates ambiguous relationships from adult and larval morphology or convergent evolution within Myctophini lineages. Paxton [
<xref ref-type="bibr" rid="B3">3</xref>
] noted that no additional characters are present within
<italic>Myctophum</italic>
to support a split between cycloid- and ctenoid-scaled
<italic>Myctophum</italic>
species. The term”near-surface” myctophids has been used for a group of myctophids represented within the genera
<italic>Loweina</italic>
,
<italic>Tarletonbeania</italic>
,
<italic>Gonichthys</italic>
,
<italic>Centrobranchus</italic>
,
<italic>Symbolophorus</italic>
and
<italic>Myctophum</italic>
with most species showing DVM behaviour and most being easily netted in surface layers at night time [
<xref ref-type="bibr" rid="B102">102</xref>
]. Denser taxon sampling within Gonichthyini, and the ctenoid-scaled Myctophini group as recognized from this study, should clarify if DVM patterns and phylogeny are correlated.</p>
</sec>
<sec>
<title>Diaphini tRNA-Met pseudogenes</title>
<p>Our included taxa in the tribe Diaphini opens up for a unique empirical case of a putative duplication event that can be observed through time for retention and deletion of various genes/pseudogenes in separate diaphinid taxa. All tRNA-Met pseudogenes were retained with 64–68 base pairs whereas other INC-regions between the tRNA genes in this segment (grey areas in Figure 
<xref ref-type="fig" rid="F6">6</xref>
) seem to be in the process of being removed. Phylogenetic congruence between the mitogenomic sequences and the diaphinid pseudogenes (Figures 
<xref ref-type="fig" rid="F5">5</xref>
and
<xref ref-type="fig" rid="F6">6</xref>
, respectively) is clear despite the short pseudogene sequence lengths. Only one other vertebrate group, scarrid parrotfishes, shows a similar pattern of gene rearrangement of the IQM-region and retention of the pseudogene [
<xref ref-type="bibr" rid="B103">103</xref>
], although the pattern of retention and deletion of INC-regions in parrotfishes is much less pronounced (see Additional file
<xref ref-type="supplementary-material" rid="S5">5</xref>
). We note that the tribe Electronini and other taxa within the subfamily Myctophinae exhibit gene rearrangements, albeit different in organization, associated with this particular region (Figure 
<xref ref-type="fig" rid="F3">3</xref>
, see Additional file
<xref ref-type="supplementary-material" rid="S4">4</xref>
). We acknowledge the missing discussion of this region in the subfamily Myctophinae; however, our results concerning this particular region in Myctophinae awaits increased taxon sampling. Topology testing using the AU-test could not reject a Notolychnini-Diaphini relationship (
<italic>P</italic>
=0.407; Table 
<xref ref-type="table" rid="T2">2</xref>
) in contrast to morphology, mitogenomic sequences and our NJ-analyses of the INC-regions (Figures 
<xref ref-type="fig" rid="F2">2</xref>
,
<xref ref-type="fig" rid="F5">5</xref>
and
<xref ref-type="fig" rid="F6">6</xref>
, respectively).</p>
<p>We note a third case of various anuran putative duplication events of the tRNA-Met gene [
<xref ref-type="bibr" rid="B104">104</xref>
] and although this case is reminiscent of the low sequence similarity between the two detected tRNA-Met genes as seen in
<italic>Notolychnus</italic>
, the tRNA-Met anticodon is repeatedly detected in anurans making this situation different to lantern- and parrotfishes.</p>
</sec>
<sec>
<title>Mt gene order rearrangements in vertebrates</title>
<p>Observations of unique gene rearrangements in Myctophidae, combined with their utility as clade defining synapomorphies, are quite numerous compared to similar findings within other vertebrate groups [
<xref ref-type="bibr" rid="B105">105</xref>
]. It should be noted that whereas mt sequences and gene order rearrangements resolve myctophiform relationships in a highly corroborative manner in this study (Figure 
<xref ref-type="fig" rid="F5">5</xref>
), taxon sampling and unknown selection on gene order and INC-sequences are problematic when inferring phylogeny. Clearly, gene order rearrangement hotspots are present in vertebrate mt genomes and selective constraints could plausibly show convergent evolution to be the reason in some cases [
<xref ref-type="bibr" rid="B106">106</xref>
]. Almost all gene order rearrangements and INC-regions presented for Myctophidae have also been found within other vertebrate groups. For example, the INC-sequence present between ATP6 and CO3 is found within
<italic>Gonostoma gracile</italic>
(order Stomiiformes) [
<xref ref-type="bibr" rid="B45">45</xref>
] and in various deep-sea anglerfishes [
<xref ref-type="bibr" rid="B47">47</xref>
]. Birds (class Aves) all have a clade-defining rearranged gene order encompassing a fragment ranging from ND6 to the CR [
<xref ref-type="bibr" rid="B107">107</xref>
], although multiple variations have subsequently occurred on top of this synapomorphy [
<xref ref-type="bibr" rid="B40">40</xref>
]. Plethodontid salamanders show multiple gene rearrangements with likely concerted evolution on duplicated regions [
<xref ref-type="bibr" rid="B108">108</xref>
]. The latter also suggested an INC-sequence between tRNA-Thr and -Pro possibly functioning as an alternative initiation site for replication. All myctophiforms successfully determined for this mt site show an INC-sequence between tRNA-Thr and -Pro, although the length of this spacer in myctophids is highly variable (see Additional file
<xref ref-type="supplementary-material" rid="S4">4</xref>
). Bakke et al. [
<xref ref-type="bibr" rid="B109">109</xref>
] postulated a selective advantage as a protein-binding site for this spacer, supported by a conservative motif sequence in codfishes. Concerted evolution on CR-duplicates is also reported from snakes [
<xref ref-type="bibr" rid="B110">110</xref>
] and mantellid frogs [
<xref ref-type="bibr" rid="B111">111</xref>
], with the latter strongly indicating recombination associated with the CR in various taxa exhibiting duplication events. Varanids and monitors show events in the variable region upstream of the CR [
<xref ref-type="bibr" rid="B112">112</xref>
] and acrodont lizards show a QIM gene order [
<xref ref-type="bibr" rid="B113">113</xref>
]. As mentioned, scarid parrotfishes all have an IMQ-gene order showing similar patterns as found in Diaphini, with an INC-sequence (pseudogene) retained upstream the ND2 gene from a putative duplication of tRNA-Met [
<xref ref-type="bibr" rid="B103">103</xref>
]. The latter suggested that the tRNA-Met pseudogene neighbouring ND2 retained a function as punctuation mark for ND2 mRNA processing. Mt tRNA transcriptional efficiency in relation to distance of initiation of replication was explored by Satoh et al. [
<xref ref-type="bibr" rid="B114">114</xref>
], who found support for highly expressed genes positioned closer to the control region. Selection on INC-regions in relation to bordering genes is an important theoretical caveat when using INC-regions as phylogenetic markers. Satoh et al. [
<xref ref-type="bibr" rid="B48">48</xref>
] initiated mt gene order comparisons within codfishes (Gadiformes), showing rearranged gene orders and INC-regions in a possible similar fashion to Myctophidae. Most notably one species,
<italic>Bathygadus antrodes,</italic>
possessed all of the gadiform rearrangements although somewhat differently arranged from other subclades. This indicates early rearrangement events subsequently modified into presently observed synapomorphic gene orders for specific gadiform clades. The most basal branching in Myctophidae,
<italic>Notolychnus</italic>
, shows mt rearrangements such as two tRNA-Met genes and a comparatively large INC-sequence between ATP6 and CO3. These features are either modified or lost in other myctophids. On the other hand, several rearranged gene orders found in various myctophid tribes are not traceable in
<italic>Notolychnus.</italic>
Gene order rearrangements within Myctophidae provide a unique example of the phylogenetic utility of mt gene orders and the problems associated with this group of markers.</p>
</sec>
</sec>
<sec sec-type="conclusions">
<title>Conclusions</title>
<p>Mitogenomes from myctophiform fishes provide a unique empirical case of mt DNA sequences and gene order rearrangements that corroborate evolutionary history. Future mitogenomic determination of the 200+ remaining species within Myctophidae will most certaninly increase our knowledge concerning issues associated with gene order rearrangement events and their utility as phylogenetic markers. Multiple different gene orders found throughout the mt genome, utility as phylogenetic markers and the unknown causes and mechanisms associated with gene duplication and subsequent retention and/or removal of sequences, calls for the determination of complete mt genomes over single genes. Our results support the use of osteology and photophores in phylogeny of Myctophiformes, the latter being a very unique feature of any deep-sea group of organisms. Larval ontogeny is also informative albeit difficult to use on higher phylogenetic levels and particularly within the subfamily Lampanyctinae.</p>
</sec>
<sec>
<title>Competing interests</title>
<p>The authors declare that they have no competing interests.</p>
</sec>
<sec>
<title>Authors’ contributions</title>
<p>All authors contributed in preparation of the study. JYP performed molecular lab work, initial analyses and drafted the original manuscript. DR, HT and TPS continually reviewed and discussed lab-procedures. JYP and EW finalized the analyses. JYP, IB, EW and MM finalized the manuscript. All authors read and approved the final manuscript.</p>
</sec>
<sec sec-type="supplementary-material">
<title>Supplementary Material</title>
<supplementary-material content-type="local-data" id="S1">
<caption>
<title>Additional file 1</title>
<p>
<bold>Table T2.</bold>
Partitioning schemes used in the present study suggested by PartitionFinder.</p>
</caption>
<media xlink:href="1471-2148-13-111-S1.pdf">
<caption>
<p>Click here for file</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="S2">
<caption>
<title>Additional file 2</title>
<p>
<bold>Saturation plots of pairwise distances of 1st, 2nd and 3rd codon positions in the 12 protein coding genes employed in the analyses.</bold>
Vertical axis shows p-distance for each codon position against total p-distance (horizontal axis). Saturation from RY-coding of the 3rd codon positions included (only transversions valued) although not used in the present study from early onset of saturation.</p>
</caption>
<media xlink:href="1471-2148-13-111-S2.pdf">
<caption>
<p>Click here for file</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="S3">
<caption>
<title>Additional file 3: Figure S1</title>
<p> Secondary structure of INC-regions. Sequence alignments and secondary tRNA structures of INC-regions are shown for synapomorphic spacers pertaining to gene order rearrangements 1, 3, 5 and 8 presented in Figure 
<xref ref-type="fig" rid="F3">3</xref>
. The remaining INC-regions presented in Figure 
<xref ref-type="fig" rid="F3">3</xref>
are relatively short sequences and are not included.</p>
</caption>
<media xlink:href="1471-2148-13-111-S3.pdf">
<caption>
<p>Click here for file</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="S4">
<caption>
<title>Additional file 4: Table T1</title>
<p> Base pair details of INC-regions found in Myctophidae. Eight mt regions correspond to gene order and INC-region synapomorphies presented in Figure 
<xref ref-type="fig" rid="F3">3</xref>
with slash (/) denoting bordering genes. The last column includes mt information from the polymorphic region from tRNA-Thr and downstream towards the control region (CR). Numbers of INC-sequence base pairs are parenthesized next to flanking genes. Greater-than sign (>) and hyphen (−) denote partial and no sequence, respectively, from failure to complete the sequences.</p>
</caption>
<media xlink:href="1471-2148-13-111-S4.pdf">
<caption>
<p>Click here for file</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="S5">
<caption>
<title>Additional file 5</title>
<p>
<bold>IMQM</bold>
<sub>
<bold>ψ</bold>
</sub>
<bold> gene order in a clade of scarid parrotfishes.</bold>
Taxa constituting a monophyletic clade in Scaridae all show similar patterns in the IMQ-region as observed within Diaphini. Grey areas show INC-regions with number of base pairs noted for each taxon and the arrow indicates possible gradual removal of INC-regions.</p>
</caption>
<media xlink:href="1471-2148-13-111-S5.pdf">
<caption>
<p>Click here for file</p>
</caption>
</media>
</supplementary-material>
</sec>
</body>
<back>
<sec>
<title>Acknowledgements</title>
<sec>
<title>No ethical issues</title>
<p>The authors declare that no ethical issues were present for this study.</p>
<p>JYP thanks K. Mabuchi, T. Kon, Y. Yamanoue, M. Nakatani, M. Seimiya, M. Yorifuji, S. Hayeun, M. Takada, M. Kawaguchi, T. Shuto, M. Kato, A. Kinjo, T. Nagasaki, (AORI), K. Sünksen, N. Hammeken, S. Hvass, H. Lund (GN-Pinngortitaleriffik) and all crew onboard R/V Pâmiut.</p>
<p>For providing tissue and access to vouchers we thank Center for Molecular Biodiversity Research (NSMT), M. Moku (AORI), H.J. Walker (SIO), S.W. Knudsen, M. Kragh, T. Menne, P. Møller, J. Nielsen (ZMUC), K. Hartel (MCZ), A.C. Bentley (KU), C. Kenealey (WU), A. Graham (CSIRO), G. Herrera, L. Rodriguez-Grana, (Chile & Uruguay, respectively), J. Paxton, A. Hay (AUS), K-T. Shao, L.Y-C. Liao, P-L. Lin (ASIZP), N.V. Gordeeva (IOGEN). A big thanks goes to H. Endo and N. Nakayama (BSKU) for onboard sampling for this study.</p>
<p>For general logistics we thank G. Langhelle, L. Emdal (ZMUB), L. Lindblom, K. Meland (UIB) and Rudolf Svendsen for contributing UW-photograph.</p>
<p>Two anonymous reviewers and editor provided highly constructive reviews of this manuscript before publication. Funding was provided by JSPS Research grants (17207007, 19207007, and 22370035) and Bergen University Museum Ph.D.- and travel grants for JYP.</p>
</sec>
</sec>
<ref-list>
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