The evolution of euhermaphroditism in caridean shrimps: a molecular perspective of sexual systems and systematics.

BACKGROUND: The hippolytid genus Lysmata is characterized by simultaneous hermaphroditism, a very rare sexual system among Decapoda. Specialized cleaning behavior is reported in a few pair-living species; these life history traits vary within the genus. Unfortunately, the systematics of Lysmata and the Hippolytidae itself are in contention, making it difficult to examine these taxa for trends in life history traits. A phylogeny of Lysmata and related taxa is needed, to clarify their evolutionary relationships and the origin of their unique sexual pattern. In this study, we present a molecular phylogenetic analysis among species of Lysmata, related genera, and several putative hippolytids. The analysis is based upon DNA sequences of two genes, 16S mtDNA and nuclear 28S rRNA. Phylogenetic trees were estimated using Bayesian Inference, Maximum Likelihood, and Maximum Parsimony. RESULTS: Phylogenetic analysis of 29 species of Lysmata, eight genera of Hippolytidae and two genera of Barbouriidae based on a single (16S, 28S) and combined gene approach (16S+28S) indicates that three groups of Lysmata differentiate according to antennular morphology: (1) Lysmata, having a multi-segmented accessory branch, (2) Hippolysmata (prior to Chace 1972), with a one-segmented accessory branch, and (3) a third group of Lysmata outliers, with one-segmented unguiform accessory branch, and close affinity to the genera Exhippolysmata and Lysmatella. The monophyly of the clade bearing a multi-segmented accessory branch is robust. Within the short accessory branch clade, species with specialized cleaning behaviors form a monophyletic clade, however, the integrity of the clade was sensitive to alignment criteria. Other hippolytid and barbouriid genera used in the analysis are basal to these three groups, including one displaying simultaneous hermaphroditism (Parhippolyte). The two barbouriid species occur in a separate clade, but among hippolytid taxa. CONCLUSIONS: The data support the historical morphological division of Lysmata into clades based on accessory branch morphology. The position of the "cleaner" shrimps, indicates that specialized cleaning behavior is a derived trait. The topologies of the cladograms support the monophyly of the barbouriids, but do not support their elevation to familial status. Taxa ancestral to the genus Lysmata display simultaneous hermaphroditism, suggesting that this life history trait evolved outside the genus Lysmata.

Reproductive isolation between 2 karyotypes in natural populations of the leaf beetle Chrysolina aurichalcea.

Japanese populations of the leaf beetle, Chrysolina aurichalcea, have 2 main karyotypes, Type 31 and Type 41, which greatly differ in chromosome number and form. We examined their geographical distribution and reproductive isolation in southern Hokkaido, northern Japan. Type 41 was widely distributed in the survey area, whereas Type 31 was restricted to a narrow portion. A transect across each karyotypic habitat revealed that both karyotypes co-occurred in a approximately 5 km wide zone, with few hybrids or hybrid derivatives. Assessment of the fine scale distribution showed that the 2 karyotypes do not occupy separate microhabitats within the sympatric zone. Karyotype combinations of copulating pairs in the sympatric zone showed no evidence of assortative mating according to karyotype. These findings suggest that postmating isolation between the 2 karyotypes is strong, whereas premating isolation is weak. It does not agree with results from previous studies, which reported a substantial degree of both post- and premating isolation between the karyotypes. Possible causes of the discrepancy in premating isolation are discussed.

Limited overwater dispersal and genetic differentiation of the snake-eyed skink (Cryptoblepharus nigropunctatus) in the Oceanic Ogasawara Islands, Japan.

The genetic differentiation and speciation of lizards on oceanic islands may be affected by their rate of overwater dispersal. Cryptoblepharus is one of the most geographically widespread scincid lizards throughout the Indo-Pacific and Australian regions. Cryptoblepharus nigropunctatus is the northernmost species of the genus, dwelling on several small Pacific islands. To examine the colonization history of this lizard, mitochondrial 16S rDNA and D-loop sequences were compared among populations of the Ogasawara Islands consisting of four island groups (the Muko-jima, Chichi-jima, Haha-jima, and Kazan groups), and an isolated island, Minamitori-shima (Marcus Island). These four groups and Minamitori-shima have not been connected to each other because each is surrounded by deep sea (>100 m). DNA analyses showed that the lizard populations on individual islands had each representative haplotypes. The ancestors of C. nigropunctatus probably arrived on the islands from the southern Pacific Ocean via wave dispersal and differentiated to produce the present state. They appear to have dispersed from their origin along two independent pathways: one between Kitaiwo-to (Kazan group) and the Muko-jima and Chichi-jima groups, and the other among the Minamitori-shima, Minamiiwo-to (Kazan group), and Haha-jima groups. Limited long-distance overwater dispersal may be responsible for the genetic structure of the C. nigropunctatus populations on these oceanic islands. However, among the small islands within the same island group, D-loop haplotypes were shared and the local genetic diversity was usually high, suggesting frequent gene flow across the same group of islands.

Rapid chromosomal changes inferred from variation in mitochondrial DNA among populations of the leaf beetle Chrysolina aurichalcea (Coleoptera: Chrysomelidae) in Japan.

There are two karyotypes among Japanese populations of the leaf beetle Chrysolina aurichalcea . In "Type 31" beetles, the diploid number of chromosomes is 2n=32 in females and 31 in males, while in "Type 41" individuals, the diploid number of chromosomes is 2n=42 in females and 41 in males. Both karyotypes occur allopatrically or parapatrically in the wild, depending on location; generally, Type 41 beetles inhabit mountainous areas and Type 31 beetles occur on lowland plains. To assess genetic differentiation among C. aurichalcea populations and between the two karyotypes, we collected 606 individuals throughout Japan, determined the karyotypes of 203 individuals, and examined sequence variation in the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene for all 606 individuals. Karyotypic analysis indicated that hybrids between the two karyotypes are rare. A haplotype phylogeny shows that neither karyotype is monophyletic. These results may be explained by ancestral polymorphism and/or interkaryotype hybridization. Population genetic analysis showed that genetic divergence between the two karyotypes is relatively low, and similar to variation within karyotypes. Our data suggest that chromosomal changes occurred rapidly and recently. We also discuss the phylogeography of this species and the association between speciation and chromosomal rearrangements.

Phylogeny of the Drosophila immigrans species group (Diptera: Drosophilidae) based on Adh and Gpdh sequences.

The immigrans species group in the Drosophilinae is one of the representative species groups of Drosophila in East Asia. Although this group constitutes a significant part of the drosophilid fauna in the Old World, only a few species have been analyzed in previous molecular phylogenetic studies. To study the phylogeny of the immigrans group, we analyzed the nucleotide sequences of two nuclear genes, alcohol dehydrogenase (Adh) and glycerol-3-phosphate dehydrogenase (Gpdh), for 36 drosophilid species, including 12 species of the immigrans group. In the resultant phylogenetic trees, 10 species of the immigrans group (D. immigrans, D. formosana, D. ruberrima, D. albomicans, D. nasuta, D. neonasuta, D. pallidifrons, D. hypocausta, D. neohypocausta, D. siamana) consistently formed a clade (the immigrans group proper), although the phylogeny within this clade did not exactly correspond to the classification of species subgroups. However, D. annulipes and D. quadrilineata, both of which belong to the quadrilineata subgroup of the immigrans group, were not included in the immigrans group proper. Furthermore, we obtained the unexpected result that D. annulipes was included in a clade comprising Scaptomyza and Hawaiian Drosophila, together with D. maculinotata of the funebris group, although the phylogenetic relationships within this clade remain uncertain and need to be substantiated with further studies. Thus, according to the present study, the immigrans group is polyphyletic.

Molecular phylogeny of the Drosophila obscura species group, with emphasis on the Old World species.

BACKGROUND: Species of the Drosophila obscura species group (e.g., D. pseudoobscura, D. subobscura) have served as favorable models in evolutionary studies since the 1930's. Despite numbers of studies conducted with varied types of data, the basal phylogeny in this group is still controversial, presumably owing to not only the hypothetical 'rapid radiation' history of this group, but also limited taxon sampling from the Old World (esp. the Oriental and Afrotropical regions). Here we reconstruct the phylogeny of this group by using sequence data from 6 loci of 21 species (including 16 Old World ones) covering all the 6 subgroups of this group, estimate the divergence times among lineages, and statistically test the 'rapid radiation' hypothesis. RESULTS: Phylogenetic analyses indicate that each of the subobscura, sinobscura, affinis, and pseudoobscura subgroups is monophyletic. The subobscura and microlabis subgroups form the basal clade in the obscura group. Partial species of the obscura subgroup (the D. ambigua/D. obscura/D. tristis triad plus the D. subsilvestris/D. dianensis pair) forms a monophyletic group which appears to be most closely related to the sinobscura subgroup. The remaining basal relationships in the obscura group are not resolved by the present study. Divergence times on a ML tree based on mtDNA data are estimated with a calibration of 30-35 Mya for the divergence between the obscura and melanogaster groups. The result suggests that at least half of the current major lineages of the obscura group originated by the mid-Miocene time (~15 Mya), a time of the last developing and fragmentation of the temperate forest in North Hemisphere. CONCLUSION: The obscura group began to diversify rapidly before invading into the New World. The subobscura and microlabis subgroups form the basal clade in this group. The obscura subgroup is paraphyletic. Partial members of this subgroup (D. ambigua, D. obscura, D. tristis, D. subsilvestris, and D. dianensis) form a monophyletic group which appears to be most closely related to the sinobscura subgroup.

Genetic differentiation and cryptic speciation in natural populations of Drosophila lacertosa.

Drosophila lacertosa, an Oriental member of the robusta species group in the virilis-repleta radiation, has a wide distribution from northern India throughout China to the Far East. Phylogenetic analyses of mitochondrial ND2 gene sequences revealed two genetically significantly diverged lineages with 2.1% DNA sequence divergence. These two lineages are largely allopatric: one is mainly found in southwest China (lineage A), whereas the other ranges central and east of China to northeast Asia (lineage B). The geographic distributions of these two clades narrowly overlap near the provincial boundaries of Yunnan and Guizhou or Guangxi, probably as a consequence of population expansion and secondary contact. These two lineages were estimated to have diverged about 0.56 Mya, and the estimated time of expansion was approximately 70,000 years ago for lineage A population, and 110,000 years ago for lineage B population. The present molecular analysis, together with nearly complete reproductive isolation between those two lineages, strongly suggests that the two lineages might be incipient species.

Molecular phylogeny of the Drosophila virilis section (Diptera: Drosophilidae) based on mitochondrial and nuclear sequences.

Regardless of the well-documented virilis species group, most groups of the Drosophila virilis section have not been completely studied at molecular level since it was suggested. Therefore, phylogenetic relationships among and within species groups of the virilis section are generally unknown. In present paper, the complete mitochondrial ND2 gene and fragment of COI gene in combination with a nuclear gene, Adh coding region, were used to derive the most extensive molecular phylogeny to date for the Drosophila virilis section. A total of 111 individuals covering 61 species were sampled in this study. Novel phylogenetic findings included (1) support for the paraphyly of the melanica and robusta species group and at least two subgroups of the robusta species group, the lacertosa and okadai subgroups, were distinguished as paraphyletic taxa. In addition, (2) present results revealed the sister relationship between D. moriwakii and the robusta subgroup, conflicting with current taxonomy regarding D. moriwakii, which was shifted from the robusta species group to the melanica group. (3) In contrast to the robusta and melanica species groups, monophyly of the polychaeta species group, the angor group and the virilis group was confirmed, respectively. However, the monophyletic quadrisetata species group was resolved with uncertainty. (4) Our analyses of combined data set suggested close relationship between the quadrisetata species group and the unpublished clefta group, and the okadai subgroup is sister to the clade comprising of the quadrisetata and clefta species groups. Within the virilis section, D. fluvialis and three tropical species groups, the polychaeta group, the angor group and the repleta group, are found to branch off earlier than other ingroup taxa. This suggests that the virilis section might have originated in the Old World tropics. Besides, the derived status of the close affinities of the quadrisetata group, the clefta group, and the melanica and robusta groups is probably the result of their adaptation to forests between subtropical and cool-temperate climate. Based on the consideration of the phylogenetic placement of the species of the virilis section, we suggest that at least five independent migrations occurred from the Old World to the New World.

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Unusual evolution of interspersed repeat sequences in the Drosophila ananassae subgroup.

New repeat sequences were found in the Drosophila ananassae genome sequence. They accounted for approximately 1.2% of the D. ananassae genome and were estimated to be more abundant in genomes of its closely related species belonging to the Drosophila bipectinata complex, whereas it was entirely absent in the Drosophila melanogaster genome. They were interspersed throughout euchromatic regions of the genome, usually as short tandem arrays of unit sequences, which were mostly 175-200 bp long with two distinct peaks at 180 and 189 bp in the length distribution. The nucleotide differences among unit sequences within the same array (locus) were much smaller than those between separate loci, suggesting within-locus concerted evolution. The phylogenetic tree of the repeat sequences from different loci showed that divergences between sequences from different chromosome arms occurred only at earlier stages of evolution, while those within the same chromosome arm occurred thereafter, resulting in the increase in copy number. We found RNA polymerase III promoter sequences (A box and B box), which play a critical role in retroposition of short interspersed elements. We also found conserved stem-loop structures, which are possibly associated with certain DNA rearrangements responsible for the increase in copy number within a chromosome arm. Such an atypical combination of characteristics (i.e., wide dispersal and tandem repetition) may have been generated by these different transposition mechanisms during the course of evolution.

A novel chimeric gene, siren, with retroposed promoter sequence in the Drosophila bipectinata complex.

Retrotransposons often produce a copy of host genes by their reverse transcriptase activity operating on host gene transcripts. Since transcripts normally do not contain promoter, a retroposed gene copy usually becomes a retropseudogene. However, in Drosophila bipectinata and a closely related species we found a new chimeric gene, whose promoter was likely produced by retroposition. This chimeric gene, named siren, consists of a tandem duplicate of Adh and a retroposed fragment of CG11779 containing the promoter and a partial intron in addition to the first exon. We found that this unusual structure of a retroposed fragment was obtained by retroposition of nanos, which overlaps with CG11779 on the complementary strand. The potential of retroposition to produce a copy of promoter and intron sequences in the context of gene overlapping was demonstrated.

Replication origin of mitochondrial DNA in insects.

The precise position of the replication origin (O(R)) of mtDNA was determined for insect species belonging to four different orders (four species of Drosophila, Bombyx mori, Triborium castaneum, and Locusta migratoria, which belong to Diptera, Lepidoptera, Coleoptera, and Orthoptera, respectively). Since the free 5' ends of the DNA strands of mtDNA are interpreted as the O(R), their positions were mapped at 1-nucleotide resolution within the A + T-rich region by using the ligation-mediated PCR method. In all species examined, the free 5' ends were found within a very narrow range of several nucleotides in the A + T-rich region. For four species of Drosophila, B. mori, and T. castaneum, which belong to holometabolous insects, although the O(R)'s were located at different positions, they were located immediately downstream of a series of thymine nucleotides, the so-called T-stretch. These results strongly indicate that the T-stretch is involved in the recognition of the O(R) of mtDNA at least among holometabolous insects. For L. migratoria (hemimetabolous insect), on the other hand, none of the long stretches of T's was found in the upstream portion of the O(R), suggesting that the regulatory sequences involved in the replication initiation process have changed through insect evolution.

The mitochondrial genome of the Japanese freshwater crab, Geothelphusa dehaani (Crustacea: Brachyura): evidence for its evolution via gene duplication.

The translocation of tRNA genes has occurred frequently among multiple independent arthropod lineages. However, the mechanisms of such gene rearrangement are still unclear. In this study we sequenced the nearly complete mitochondrial genome of the Japanese freshwater crab, Geothelphusa dehaani (Decapoda, Brachyura, Potamidae). Its size is relatively large (app. 24 kb) for higher animal mtDNA and is due to the presence of many intergenic noncoding sequences (IGNs), one of which is a large repeat region (>7.0 kb). Excluding the repeat region and the putative control region (514 bp), the total size of IGNs (1953 bp) is still the largest reported in arthropods. In comparison to the proposed ancestral arrangement for the insect-crustacean clade, G. dehaani displays changes in the positional order for three tRNA genes (trnQ, trnL(uur) and trnH). At the putative former and current locations for trnQ and trnH, there are IGNs with high similarities to corresponding sequences in the ancestral arrangement. This suggests the occurrence of partial duplication, followed by a loss of function for one copy of these two duplicated genes in G. dehaani. Furthermore, three trnLs (one trnL(uur) and two trnL(cun)s) are found in G. dehaani; their sequences are highly similar to each other (70.8-82.5%) and similar to the trnL(cun) of other arthropods. This suggests that duplication of trnL(cun) occurred at least twice, and the current trnL(uur) of G. dehaani arose by a subsequent anticodon alternation. These observations provide direct evidence for the "duplication-random loss" model for mtDNA gene rearrangement. Comparisons with other available Brachyuran data(http://www.ncbi.nlm.nih.gov/genomes/ORGANELLES/6657.html) suggest that two of the three tRNA translocations are unique in G. dehaani. For G. dehaani, where duplications have occurred but deletion of extra sequences is incomplete, the IGNs found at the ancestral or trans-locations are likely to be footprints of relatively recent mitochondrial genome evolution.

The Drosophila obscura species-group (Diptera, Drosophilidae) from Yunnan Province, Southern China.

Three new and two known species of the Drosophila (Sophophora) obscura species-group are reported from Yunnan Province, southern China. The sinobscura species-subgroup is newly established by D. sinobscura, D. hubeiensis and D. luguensis sp. nov. Geographic distribution of the obscura group in and around China is discussed, and a key to 10 Chinese species of the obscura group is provided.

Cloning of the genes for the pituitary glycoprotein hormone alpha and follicle-stimulating hormone beta subunits in the Japanese crested ibis, Nipponia nippon.

We have isolated a part of the gene for the pituitary glycoprotein hormone common alpha subunit (PGHalpha) and the whole gene for the follicle-stimulating hormone beta subunit (FSHbeta) in the Japanese crested ibis (Nipponia nippon), a critically endangered bird species in East Asia. The nucleotide sequence of a part of the PGHalpha gene (5026 bp) contained three exons holding the whole coding and 3' untranslated regions, but lacked a 5' untranslated region. Its exon-intron structure was similar to that in mammals, but different from that in teleosts in the location of the second intron. For the FSHbeta gene, the nucleotide sequence of 7633 bp was assembled from two phage clones. The exon-intron structure of three exons and two introns was similar to that observed in mammals and teleosts. In the putative promoter region of the ibis FSHbeta gene, a progesterone responsive element (PRE)-like sequence and two AP-1 responsive element-like sequences reported in the ovine FSHbeta gene were not conserved in complete form. The increased number of ATTTA motifs in the putative 3' untranslated region in comparison with those in Japanese quail and chicken FSHbeta cDNA suggested that more rapid degradation of FSHbeta mRNA occurs in this species. Deduced amino acid sequences of the ibis PGHalpha and FSHbeta showed high similarities with those of the corresponding subunits of other avian species. This is the first report on the genomic sequences of the PGHalpha and FSHbeta in an avian species.

Cloning of the gene for the thyrotropin beta subunit in the Japanese crested ibis, Nipponia nippon.

We isolated a putative gene for the thyrotropin beta subunit (TSHbeta) from two types of genomic libraries of the Japanese crested ibis, Nipponia nippon. Exon-intron structure was deduced by comparing the determined sequence with those of TSH beta cDNA of other birds. The deduced amino acid sequence shows extensive similarities to those of the other birds, which assures our assumption that the acquired nucleotide sequence represents the TSHbeta gene. The assembled genomic fragment is 4192 bp in size and consists of 1937 bp of putative 5' flanking region followed by exon-intron structure with three exons and two introns, similar to those observed in rat, human and goldfish counterparts. Locations of introns are also similar to those in mammals and goldfish. Comparison of the 5' flanking region of the ibis TSHbeta gene with those of mammals reveals that several regulatory sequences, such as negative thyroid hormone responsive element (nTRE), Pit-1 responsive element, and AP-1 responsive element, which were characterized in mammalian TSHbeta genes, are also found in the promoter region. This is the first report on the exon-intron structure and 5' flanking region of the TSHbeta gene in an avian species.

Karyotype differentiation and reproductive isolation among natural populations of Drosophila lacertosa.

Drosophila lacertosa is widely distributed from northern India to Far East of Russia throughout China. We have studied geographical distributions of three kinds of chromosomal karyotypes, type D (2n=10, 4 pairs of V-shaped metacentric chromosomes and a pair of micro-chromosomes), type L (2n=10, 5V with a pair of large-sized heterochromatic autosomes) and type M (2n=10, 5V with middle-sized ones). Type D was found exclusively in local populations of D. lacertosa distributed in Yun-Gui Plateau, southwestern China. Both type L and M have a wide range of distribution, and the former occurred in subtropical regions of China including Taiwan Island, whereas the latter in cool temperate regions of East Asia covering Far East of Russia, Korea and the Japan Islands. A strong pre-mating isolation was detected between flies with type D and those with type L or M. These data demonstrate that genetic differentiation leading to cryptic speciation might have occurred in natural populations of Drosophila lacertosa.