Mitochondrial DNA in Atherina (Teleostei,Atheriniformes): differential distribution of an intergenic spacer in lagoon and marine forms of Atherina boyeri

The big‐scale sand smelt Atherina boyeri lives in fresh water, brackish water and sea water of the western Atlantic Ocean and Mediterranean Sea. Previous studies concerning distribution, biometric characters and genetic molecular markers have suggested the possible existence of two or even three different groups or species of sand smelt, one ‘lagoon’ type and one (or two – punctuated and non‐punctuated on the flanks) ‘marine’ type. In this study, the presence and the localization of an insertion was described, c. 200 bp in length, in the mtDNA of the lagoon and marine punctuated specimens of A. boyeri and its absence in the marine non‐punctuated specimens, as well as in other two congeneric species, Atherina hepsetus and Atherina presbyter, and in the atheriniform Menidia menidia. The intergenic spacer is located between the tRNA^Glu and cytochrome b (cyt b) genes and shares a c. 50% sequence similarity with cyt b. The distribution and the features of the intergenic spacer suggest that it might have originated from an event of gene duplication, which involved the cyt b gene (or, more likely, a part of it) and which took place in the common ancestor of the lagoon and the marine punctuated specimens. The data obtained therefore support the hypothesis of the existence of three cryptic and, or sibling species within the A. boyeri taxon and provide a genetic molecular marker to distinguish them.     

Molecular phylogenetics of slit‐faced bats (Chiroptera: Nycteridae) reveal deeply divergent African lineages

The bat family Nycteridae contains only the genus Nycteris, which comprises 13 currently recognized species from Africa and the Arabian Peninsula, one species from Madagascar, and two species restricted to Malaysia and Indonesia in South‐East Asia. We investigated genetic variation, clade membership, and phylogenetic relationships in Nycteridae with broad sampling across Africa for most clades. We sequenced mitochondrial cytochrome b (cytb) and four independent nuclear introns (2,166 bp) from 253 individuals. Although our samples did not include all recognized species, we recovered at least 16 deeply divergent monophyletic lineages using independent mitochondrial and multilocus nuclear datasets in both gene tree and species tree analyses. Mean pairwise uncorrected genetic distances among species‐ranked Nycteris clades (17% for cytb and 4% for concatenated introns) suggest high levels of phylogenetic diversity in Nycteridae. We found a large number of designated clades whose members are distributed wholly or partly in East Africa (10 of 16 clades), indicating that Nycteris diversity has been historically underestimated and raising the possibility that additional unsampled and/or undescribed Nycteris species occur in more poorly sampled Central and West Africa. Well‐resolved mitochondrial, concatenated nuclear, and species trees strongly supported African ancestry for SE Asian species. Species tree analyses strongly support two deeply diverged subclades that have not previously been recognized, and these clades may warrant recognition as subgenera. Our analyses also strongly support four traditionally recognized species groups of Nycteris. Mitonuclear discordance regarding geographic population structure in Nycteris thebaica appears to result from male‐biased dispersal in this species. Our analyses, almost wholly based on museum voucher specimens, serve to identify species‐rank clades that can be tested with independent datasets, such as morphology, vocalizations, distributions, and ectoparasites. Our analyses highlight the need for a comprehensive revision of Nycteridae.     

Saurogobio punctatus sp. nov., a new cyprinid gudgeon (Teleostei: Cypriniformes) from the Yangtze River,based on both morphological and molecular data

A new cyprinid gudgeon, Saurogobio punctatus sp. nov., is described based on specimens collected from the Yangtze River, China. The new species can be distinguished from its congeners by differences in both morphology and the cytochrome b (cytb) gene sequence. Numerous minute blackish spots are scattered on dorsal and caudal fins in S. punctatus sp. nov. v. absent in the other seven valid Saurogobio species. The new species can be further distinguished from its congeners by the following unique combination of characters: a dorsal fin with eight branched rays; absence of scales in chest area before pectoral origin; upper and lower lips thick, covered with papillae; and a papillose mental pad approximately triangular. Morphologically, the new species most resembles the Chinese lizard gudgeon Saurogobio dabryi, but the new species lays yellowish adhesive eggs v. white pelagic eggs in S. dabryi. A phylogenetic analysis of all Saurogobio species based on cytb gene sequences indicated that S. punctatus sp. nov was distinctly separated from its congeners, with mean sequence divergence ranging from 12·6 to 21·0%. Therefore, molecular data further supported the distinctiveness of the new species.     

Morphological and molecular variation in the least madtom Noturus hildebrandi (Siluriformes: Ictaluridae), a Mississippi Embayment endemic: evidence for a cryptic lineage in the Hatchie River

Sheared principal component analysis of 40 morphometric characteristics measured for 146 individuals and relative frequencies of pigmentation patterns scored for 980 individuals of the least madtom Noturus hildebrandi, a diminutive catfish endemic to eastern lowland drainages of the Mississippi Embayment region of North America, suggested a clinal pattern of morphological variation extending across the range from north to south. DNA sequence data representing 90 individuals from the mitochondrial gene cytochrome b (cytb) analysed using Bayesian phylogenetic methods recovered four major haplotype clades, suggestive of a high degree of isolation by drainage. Individual gene trees of cytb and four additional nuclear loci as well as trees based on concatenated datasets of these genes consistently recovered a cryptic lineage of individuals from the Hatchie River drainage that is morphologically indistinguishable from surrounding populations. Gene‐tree analyses failed to recover a monophyletic N. hildebrandi with respect to Noturus baileyi. A coalescence‐based species tree analysis, however, did recover N. hildebrandi monophyly with high support, suggesting that relationships reflected in individual gene trees and concatenated datasets are in part artefacts of incomplete lineage sorting or an ancient introgressive event. Results are consistent with the hypothesis of an ancient connection between the Hatchie and Tennessee River systems. Current subspecific designations are of limited utility as they reflect morphological variation and are not entirely consistent with phylogeny. Discrepancies between the pattern of variation observed in the morphological and molecular data may be explained by recent local adaptation to individual stream conditions that masks deeper evolutionary divergences.     

Assessing the diversity and distribution of Cephalothrix species (Nemertea: Palaeonemertea) in European waters by comparing different species delimitation methods

Soft‐bodied marine taxa, like ribbon worms (Nemertea), often lack clear diagnostic morphological characters impeding traditional species delimitation. Therefore, recent studies concentrated on molecular genetic methods to solve taxonomic issues. Different delimitation methods were employed to explore species boundaries and the presence of cryptic species. However, the performance of the different delimitation methods needs to be tested. A particularly promising nemertean genus in this regard is the palaeonemertean genus Cephalothrix that is commonly found in European waters. In order to gain information on the number and distribution of European cephalotrichids and to test different tree‐based and non‐tree‐based delimitation methods, we analyzed a dataset comprising the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) of 215 European Cephalothrix specimens, of which 78 were collected for this study. Our results show the presence of 12–13 European lineages of which several can be assigned to known European species. Analyzing a second dataset comprising 74 additional sequences from the Pacific and the Atlantic Oceans helped identify some of the unassigned European specimens. One resulting clade seems to represent a non‐native introduced Cephalothrix species, while another has never been recorded from Europe before. In our analysis, especially the tree‐based methods and the phylogenetic analysis proved to be a useful tool when delimiting species. It remains unclear whether the different identified clades result from cryptic speciation or from a high genetic variability of the COI gene.     

Evaluating the utility of cox1 for cetacean species identification

The mitochondrial cytochrome c oxidase I (cox1) gene has been promoted as a universal reference gene, or barcode, to identify organisms to the species level. We evaluated whether cox1 would be appropriate to diagnose cetacean species. The 5′ end of cox1 (686 base pairs, bp) was sequenced for 46 of 86 recognized species of cetaceans. In addition, we included 105 sequences from GenBank, increasing our taxonomic coverage to 61 species. Particular focus was placed on sampling two subfamilies that contain closely related taxa: the Delphininae and the Globicephalinae. Species‐specific sequences were observed for all but three taxa (Delphinus delphis, D. capensis, and Stenella coeruleoalba). Although correct assignment was seen for most species, significant overlap between intra‐ and interspecific variation makes cox1 an imperfect barcode for cetaceans. The efficacy of cox1 was compared to the 5′ end of the cytochrome b (cytb) gene, a mitochondrial region routinely used for cetacean species identification. Although cytb performed better than cox1 for some species, this marker could not differentiate other closely related taxa (Eubalaena spp.). Species identification for taxa not reliably identified using cox1 or cytb might be best addressed through use of multiple mitochondrial DNA fragments or other newly developed markers.     

Insight into an island radiation: the Tarentola geckos of the Cape Verde archipelago

Aim To reassess the relationships between Tarentola geckos from the Cape Verde Islands by including specimens from all islands in the range. To determine the variation within forms by sequencing over 400 specimens, thereby allowing the discovery of cryptic forms and resolving some of the issues raised previously. This extensive sampling was also used to shed light on distributions and to explain genetic diversity by comparing the ages and ecological and geological features of the islands (size, elevation and habitat diversity). Location The Cape Verde Islands: an oceanic archipelago belonging to the Macaronesian biogeographic region, located around 500 km off Senegal. Methods A total of 405 new specimens of Tarentola geckos were collected from nine islands with very different geological histories, topography, climate and habitats. Mitochondrial cytochrome b (cyt b) gene and 12S rRNA partial sequences were obtained and analysed using phylogenetic methods and networks to determine molecular diversity, demographic features and phylogeographic patterns. Results The phylogenetic relationships between all known forms of Cape Verdean Tarentola specimens were estimated for the first time, the relationships between new forms were assessed and previously hypothesized relationships were re‐examined. Despite the large sample size, low intraspecific diversity was found using a 303‐bp cyt b fragment. Star‐like haplotype networks and statistical tests suggest the past occurrence of a rapid demographic and geographical expansion over most of the islands. Genetic variability is positively correlated with size, elevation and habitat diversity of the islands, but is not linearly related to the age of the islands. Biogeographical patterns have, in general, high concordance with phylogenetic breaks and with the three eco‐geographical island groups. Volcanism and habitat diversity, both tightly linked with island ontogeny, as postulated by the general dynamic model of oceanic island biogeography, as well as present and historical size of the islands appear to be the main factors explaining the genetic diversity of this group. Main conclusions The Tarentola radiation was clarified and is clearly associated with the geological and ecological features of the islands. Two factors may account for the low intraspecific variation: (1) recent volcanic activity and high ecological stress, and (2) poor habitat diversity within some islands. More studies are needed to align taxonomy with phylogenetic relationships, whereas GIS modelling may help to predict precise species distributions.     

Macroevolutionary relationships of species of Drosophila melanogaster group based on mtDNA sequences

The phylogenetic relationships among the Drosophila melanogaster group species were analyzed using approximately 1700 nucleotide-long sequences of the mitochondrial DNA. Phylogenetic analysis was performed using this region consisting of a part of the cytochrome b (cytb) coding gene, the entire coding sequences of tRNA-Leu, tRNA-Ser and the first subunit of NADH dehydrogenase (NADH1), and a part of the 16S-rRNA gene. The study of these sequences showed that this region of mtDNA is very invariable, as regards with the type of the genes that it contains, as well as the order that they are located on it. The resulting phylogenetic trees reveal a topology that separates the species into three main ancestral lines, leading to the following subgroups: (a) ananassae subgroup, (b) montium subgroup, and (c) melanogaster and Oriental subgroups. The inferred topology complements and generally agrees with previously proposed classifications based on morphological and molecular data.     

Testing phylogenetic hypotheses for reconstructing the evolutionary history of Dolichopoda cave crickets in the eastern Mediterranean

Aim To investigate the molecular phylogenetic divergence and historical biogeography of cave crickets belonging to the genus Dolichopoda (Orthoptera, Rhaphidophoridae). Location Caves in continental and insular Greece. Methods We sequenced 1967 bp of mitochondrial DNA, corresponding to three fragments of the small and large subunit of the ribosomal RNA (16S and 12S rRNA, respectively) and to the subunit I of cytochrome oxidase (COI), to reconstruct phylogenetic relationships among all 30 known Greek species of Dolichopoda. Alternative hypotheses about the colonization of the Hellenic Peninsula by Dolichopoda species were tested by comparing the degree of discordance between species trees and gene trees under four plausible biogeographical scenarios. Results The present study revealed a rather well resolved phylogeny at species level, identifying a number of clades that represent long‐separated lineages and diverse evolutionary histories within the genus Dolichopoda. Two main clades were revealed within Hellenic–Aegean species, identifying a north‐western and a south‐eastern species group. Based on Bayesian analysis, we applied a relaxed molecular clock to estimate the divergence times between the lineages. The results revealed that the origins of eastern Mediterranean lineages are much older than those of previously studied western Mediterranean Dolichopoda. Tests of alternative biogeographical hypotheses showed that a double colonization of the Hellenic Peninsula, following separate continental and trans‐Aegean routes during the Messinian stage, best accounts for the present distribution of Greek Dolichopoda species. Main conclusions Reconstruction and biogeographical hypothesis testing indicated that the colonization of Greece by Dolichopoda species comprised two episodes and two different routes. The southern lineage probably arose from a trans‐Aegean colonization during the Messinian salinity crisis (5.96–5.33 Ma). The northern lineage could be the result of dispersal from the north through the Balkan Peninsula. The opening of the Mid‐Aegean Trench could have promoted an initial diversification within the uprising Anatolian Plateau, while the Messinian marine regression offered the conditions for a rapid dispersal through the whole Aegean–Hellenic region. In addition, climatic events during the Plio‐Pleistocene may have been responsible for the speciation within each of the two different phylogeographical units, principally attributable to vicariance events.     

Evidences for genetic differentiation within the highly endemic and endangered annual fish Austrolebias nigrofasciatus (Cyprinodontiformes: Rivulidae)

Samples of Austrolebias nigrofasciatus (n = 103), an endangered species of annual fish endemic to a small area of the Patos-Mirim lagoon system encompassing the São Gonçalo Channel lowlands, were collected from eight isolated temporary ponds, four located at the known distribution range of the species and four located along the Piratini River lowlands, where morphologically different individuals were found. In the laboratory, fragments of the mitochondrial cytochrome c oxidase I (coI), cytochrome b (cytb) and nuclear rhodopsin (rho) genes were amplified, purified and sequenced for 100, 99 and 58 of these individuals, respectively. Samples were further analysed using phylogenetic and phylogeographic methods to evaluate the patterns of genetic diversity and differentiation presented within and between populations, while assessing their evolutionary history, in order to guide the application of further conservation strategies. We found that the four new populations from the Piratini River lowlands encompass a different lineage of A. nigrofasciatus that diverged from that encountered in the São Gonçalo Channel at approximately 0.165 M years before present, during a population expansion and did not yet attain reciprocal monophyly. This divergence was associated with a glacial event that was preceded by an interglacial period putatively associated with the dispersal. Moreover, significant levels of genetic differentiation and a high number of exclusive haplotypes could be encountered even in micro-geographical scales, as in the comparisons between populations located within the same major lineage, indicating each of them may encompass independent management units. Conservation actions are certainly urgent, especially in the face of signs of a recent bottleneck.     

Solenicola setigera is the first characterized member of the abundant and cosmopolitan uncultured marine stramenopile group MAST‐3

Culture‐independent molecular methods based on the amplification, cloning and sequencing of small‐subunit (SSU) rRNA genes are a powerful tool to study the diversity of prokaryotic and eukaryotic microorganisms for which morphological features are not conspicuous. In recent years, molecular data from environmental surveys have revealed several clades of protists lacking cultured and/or described members. Among them are various clades of marine stramenopiles (heterokonts), which are thought to play an essential ecological role as grazers, being abundant and distributed in oceans worldwide. In this work, we show that Solenicola setigera, a distinctive widespread colonial marine protist, is a member of the environmental clade MArine STramenopile 3 (MAST‐3). Solenicola is generally considered as a parasite or an epiphyte of the diatom Leptocylindrus mediterraneus. So far, the ultrastructural, morphological and ecological data available were insufficient to elucidate its phylogenetic position, even at the division or class level. We determined SSU rRNA gene sequences of S. setigera specimens sampled from different locations and seasons in the type locality, the Gulf of Lions, France. They were closely related, though not identical, which, together with morphological differences under electron microscopy, suggest the occurrence of several species. Solenicola sequences were well nested within the MAST‐3 clade in phylogenetic trees. Since Solenicola is the first identified member of this abundant marine clade, we propose the name Solenicolida for the MAST‐3 phylogenetic group.     

Phylogenetic topology and timing of New Zealand olive shells are consistent with punctuated equilibrium

The olive shells of the genus Amalda comprises readily recognized species of marine neogastropod mollusks found around the world. The New Zealand Amalda fauna has particular notoriety as providing one of the best demonstrations of evolutionary morphological stasis, a prerequisite for punctuated equilibrium theory. An excellent fossil record includes representation of three extant endemic Amalda species used to explore patterns of form change. However, the phylogenetic relationship of the New Zealand Amalda species and the timing of their lineage splitting have not been studied, even though these would provide valuable evidence to test predictions of punctuated equilibrium. Here, we use entire mitogenome and long nuclear rRNA gene cassette data from 11 Amalda species, selected from New Zealand and around the world in light of high rates of endemicity among extant and fossil Amalda. Our inferred phylogenies do not refute the hypothesis that New Zealand Amalda are a natural monophyletic group and therefore an appropriate example of morphological stasis. Furthermore, estimates of the timing of cladogenesis from the molecular data for the New Zealand group are compatible with the fossil record for extant species and consistent with expectations of punctuated equilibrium.     

Canary Island great spotted woodpecker ( Dendrocopos major ) has distinct mtDNA

We have analyzed nucleotide sequences of the mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 2 (nd2) genes to elucidate the phylogenetic status (genetic variation) of Canary Island populations of great spotted woodpeckers (Dendrocopos major) that are on the western fringe of the distribution range. Based on these two genes, differences are found between a clade from the Canaries and the rest of the range studied. No differences were observed within the two races found on this archipelago.     

Could pseudogenes be widespread in ants? Evidence of numts in the leafcutter ant Acromyrmex striatus (Roger, 1863) (Formicidae: Attini)

The incorporation of fragments of mitochondrial DNA (mtDNA) in the nuclear genome, known as numts (nuclear mitochondrial pseudogenes), undermines general assumptions concerning the use of mtDNA in phylogenetic and phylogeographic studies. Accidental amplifications of these nuclear copies instead of the mitochondrial target can lead to crucial misinterpretations, thus the correct identification of numts and their differentiation from true mitochondrial sequences are important in preventing this kind of error. Our goal was to describe the existence of cytochrome b (cytb) numts in the leafcutter ant Acromyrmex striatus (Roger, 1863). PCR products were directly sequenced using a pair of universal primers designed to amplify the cytb gene of these insects. Other species of leafcutter ants were also sequenced. The sequences were analyzed and the numts were identified by the presence of double peaks, indels and premature stop codons. Only A. striatus clearly showed the presence of numts, while the other species displayed the expected amplification of the mtDNA cytb gene target using the same primer pair. We hope that our report will highlight the benefits and challenges of using mtDNA in the molecular phylogenetic reconstruction and phylogeographic studies of ants, while establishing the importance of numts reports for future studies.     

16S rRNA GENE HETEROGENEITY IN THE FILAMENTOUS MARINE CYANOBACTERIAL GENUS LYNGBYA1

The SSU (16S) rRNA gene was used to investigate the phylogeny of the cyanobacterial genus Lyngbya as well as examined for its capacity to discriminate between different marine species of Lyngbya. We show that Lyngbya forms a polyphyletic genus composed of a marine lineage and a halophilic/brackish/freshwater lineage. In addition, we found morphological and genetic evidence that Lyngbya spp. often grow in association with other microorganisms, in particular smaller filamentous cyanobacteria such as Oscillatoria, and propose that these associated microorganisms have led to extensive phylogenetic confusion in identification of Lyngbya spp. At the species level, the phylogenetic diversity obtained from the comparison of 16S rRNA genes exceeded morphological diversity in Lyngbya. However, the expectation that this improved phylogeny would be useful to species and subspecies identification was eliminated by the fact that phylogenetic species did not correlate in any respect with the species obtained from current taxonomic systems. In addition, phylogenetic identification was adversely affected by the presence of multiple gene copies within individual Lyngbya colonies. Analysis of clonal Lyngbya cultures and multiple displacement amplified (MDA) single‐cell genomes revealed that Lyngbya genomes contain two 16S rRNA gene copies, and that these typically are of variable sequence. Furthermore, intragenomic and interspecies 16S rRNA gene heterogeneity was approximately of the same magnitude. Hence, the intragenomic heterogeneity of the 16S rRNA gene overestimates the microdiversity of different strains and does not accurately reflect speciation within cyanobacteria, including the genus Lyngbya.     

Mitochondrial DNA Variation, Phylogenetic Relationships, and Evolution of Four Mediterranean Genera of Soles (Soleidae, Pleuronectiformes)

To increase knowledge about the systematics and evolution of Mediterranean soles, we assessed mitochondrial DNA variation, molecular phylogeny, and evolution in eight species from the genera Solea, Microchirus, Monochirus, and Buglossidium by large ribosomal subunit (16S) and cytochrome b (cytb) sequence analysis. Relevant molecular features are the great variation of base composition among species at the third codon in cytb and the heterogeneity of the nucleotide substitution rate. Phylogenies recovered using 16S nucleotide and cytb amino acid sequences agree with those based on morphology in assessing monophyly of Solea species and ancestry of Buglossidium luteum, but they are against the intergeneric differentiation of Microchirus and Monochirus. Conversely, phylogenetic trees based on cytb nucleotide sequences yielded relationships among taxa regardless of their evolutionary histories. The incongruities between morphological and molecular issues suggest the need for reassessing the systematic value of some morphological characters. Approximate estimates of the divergence time of Mediterranean soleid lineages range from 40 to 13 Mya (Oligocene–Miocene), indicating an ancient origin for the group. Received August 31, 1999; accepted December 17, 1999.     

Variation of cytochrome b gene in sympatric chars from kronotsky lake (Kamchatka Peninsula)

In previously identified sympatric char forms (species) from Kronotsky Lake (Kamchatka Peninsula) and a number of other chars from the genus Salvelinus, nucleotide sequences of the mtDNA cytochrome b gene were determined and phylogenetic analysis of these chars was performed. The divergence estimate of the cytochrome b gene in the Kronotsky char group (0.48%) coincided with the interpopulation difference estimates in northern Dolly Varden, which indicates a relatively recent divergence of the chars from Kronotsky Lake. Haplotypes of each form of the Kronotsky Lake chars are divided into two groups, which belonged to two different phylogenetic mtDNA lineages of northern Dolly Varden from Chukotka and Kamchatka. The formation of the Kronotsky char forms proved to be unrelated to the observed variability of the mtDNA cytochrome b gene.     

Geometric morphometrics as a tool for identifying emperor fish (Lethrinidae) larvae and juveniles

The aim of this study was to evaluate the efficiency of geometric morphometrics for describing the body shape of fish larvae and juveniles, and identifying them to species, in comparison with traditional linear measurements. Species of emperor fishes (Perciformes: Lethrinidae, genus Lethrinus) were chosen as the model group, as the late larval and early juvenile stages in this genus are particularly difficult to identify. Forty‐five individuals of different species of Lethrinus were collected from the south‐western lagoon of New Caledonia between May 2005 and March 2006. The individuals were first identified to species by their partial cytochrome‐b gene sequence. They were then morphologically characterized using eight linear measurements and 23 landmarks recorded on digital photographs. Except for a small proportion of individuals, geometric morphometrics gave better results to distinguish the different species than linear measurements. A ‘leave one out’ approach confirmed the nearly total discrimination of recently settled Lethrinus genivittatus and Lethrinus nebulosus, whereas traditional identification keys failed to distinguish them. Therefore, geometric morphometrics is a promising tool for identifying fish larvae and juveniles to species. An effective approach would require building image databases of voucher specimens associated with their DNA barcodes. These images could be downloaded by the operator and processed with the specimens to be identified.     

Genetic Variations in Two Seahorse Species (Hippocampus mohnikei and Hippocampus trimaculatus): Evidence for Middle Pleistocene Population Expansion

Population genetic of seahorses is confidently influenced by their species-specific ecological requirements and life-history traits. In the present study, partial sequences of mitochondrial cytochrome b (cytb) and control region (CR) were obtained from 50 Hippocampus mohnikei and 92 H. trimaculatus from four zoogeographical zones. A total of 780 base pairs of cytb gene were sequenced to characterize mitochondrial DNA (mtDNA) diversity. The mtDNA marker revealed high haplotype diversity, low nucleotide diversity, and a lack of population structure across both populations of H. mohnikei and H. trimaculatus. A neighbour-joining (NJ) tree of cytb gene sequences showed that H. mohnikei haplotypes formed one cluster. A maximum likelihood (ML) tree of cytb gene sequences showed that H. trimaculatus belonged to one lineage. The star-like pattern median-joining network of cytb and CR markers indicated a previous demographic expansion of H. mohnikei and H. trimaculatus. The cytb and CR data sets exhibited a unimodal mismatch distribution, which may have resulted from population expansion. Mismatch analysis suggested that the expansion was initiated about 276,000 years ago for H. mohnikei and about 230,000 years ago for H. trimaculatus during the middle Pleistocene period. This study indicates a possible signature of genetic variation and population expansion in two seahorses under complex marine environments.