Phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs have been determined by geological processes and climate change in the Late Cenozoic

AIM: Our aims were to assess the phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs and to estimate divergence times using different geological scenarios. We related divergence times to past geological events and discuss the relevance of our data for the systematics of eastern Mediterranean water frogs. LOCATION: The eastern Mediterranean region. METHODS: Genetic diversity and divergence were calculated using sequences of two protein-coding mitochondrial (mt) genes: ND2 (1038 bp, 119 sequences) and ND3 (340 bp, 612 sequences). Divergence times were estimated in a Bayesian framework under four geological scenarios representing alternative possible geological histories for the eastern Mediterranean. We then compared the different scenarios using Bayes factors and additional geological data. RESULTS: Extensive genetic diversity in mtDNA divides eastern Mediterranean water frogs into six main haplogroups (MHG). Three MHGs were identified on the Anatolian mainland; the most widespread MHG with the highest diversity is distributed from western Anatolia to the northern shore of the Caspian Sea, including the type locality of Pelophylax ridibundus. The other two Anatolian MHGs are restricted to south-eastern Turkey, occupying localities west and east of the Amanos mountain range. One of the remaining three MHGs is restricted to Cyprus; a second to the Levant; the third was found in the distribution area of European lake frogs (P. ridibundus group), including the Balkans. MAIN CONCLUSIONS: Based on geological evidence and estimates of genetic divergence we hypothesize that the water frogs of Cyprus have been isolated from the Anatolian mainland populations since the end of the Messinian salinity crisis (MSC), i.e. since c. 5.5-5.3 Ma, while our divergence time estimates indicate that the isolation of Crete from the mainland populations (Peloponnese, Anatolia) most likely pre-dates the MSC. The observed rates of divergence imply a time window of c. 1.6-1.1 million years for diversification of the largest Anatolian MHG; divergence between the two other Anatolian MHGs may have begun about 3.0 Ma, apparently as a result of uplift of the Amanos Mountains. Our mtDNA data suggest that the Anatolian water frogs and frogs from Cyprus represent several undescribed species.     

New data on the evolution of the Cretan spiny mouse,Acomys minous (Rodentia: Murinae), shed light on the phylogenetic relationships in the cahirinus group

The karyotype of the Cretan spiny mouse Acomys minous was examined with chromosome banding techniques in 53 individuals from 12 localities of Crete, aiming to gain a more detailed knowledge on the chromosomal constitution and variability of its natural populations. We found that it consists of three Robertsonian (Rb) populations with 2n = 38, 2n = 40 and 2n = 42, respectively, the last one being reported for the first time, and with stable fundamental number (FNa = 66, FN = 68). The G‐banding pattern proves that the Rb populations are closely linked phylogenetically by the many common Rb fusions and the lack of monobrachial homologies. In addition, they appear to freely mate at their contact areas, producing viable and fertile hybrids. No other type of chromosomal rearrangement appears to have played part in the chromosomal evolution of this species, at least in the recent past, as indicated also by the study of the telomeric sequences. Heterochromatin appears to be restricted to the pericentromeric position of all acrocentric and most biarmed autosomes, as well as of the X chromosome, whereas the Y chromosome is uniformly, yet faintly heterochromatic. Chromosome banding comparison of the karyotypes in A. minous with those of the other species in the cahirinus group (i.e. Acomys cahirinus, Acomys cilicicus, and Acomys nesiotes) proves their very close phylogenetic relationship, further reinforced by the study of the cytochrome b sequences, and that A. minous possesses the ancestral karyotype of the group. It is suggested that at least two of the karyotypes that characterize A. minous today, pre‐existed in North Africa before it colonized Crete and that the specific status of the four members in the cahirinus group may need to be revisited. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 498–509.     

Phylogeography of spiny mice (genus Acomys, Rodentia: Muridae) from the south-western margin of the Sahara with taxonomic implications

The present study aims to depict the overall pattern of Acomys history in south-western Sahara. We tested the specific predictions that: (1) several mitochondrial clades can be identified and that they coincide with the described species; (2) successive phases of desert expansion and contraction during the last 3 Myr have resulted in several phases of demographic expansion and population fragmentation in Acomys ; and (3) isolation-by-distance occurs. The extent of phylogeographic patterns and molecular genetic diversity (cytochrome b gene and D-loop) were addressed in a survey of 90 individuals of Acomys from 38 localities. Our phylogeographical analyses show a strong genetic structure within western Saharan Acomys , with several phylogroups displaying non-overlapping geographic distributions. Restricted gene flow with isolation-by-distance was recorded and a signal of population expansion was detected within several clades. We suggest that during arid or semi-arid paleoclimatic periods, when large sandy areas were present, Acomys was restricted to rocky massifs, whereas, in more humid periods, when savannah and/or steppe habitat prevailed, this species was able to disperse and to survive outside rocky areas because food resources were abundant. Based on a comprehensive sampling and the use of an integrative approach (i.e. combining cytogenetic, molecular and morphological data), we firmly propose that Acomys airensis should be considered as a junior synonym of Acomys chudeaui .  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 29–46.     

Species delimitation in the Acomys cahirinus–dimidiatus complex (Rodentia, Muridae) inferred from chromosomal and morphological analyses

Our earlier chromosome banding studies of Acomys cahirinus and Acomys dimidiatus (the latter long considered to be a subspecies of the former) revealed that, despite very close diploid numbers (36 vs. 38), these taxa possess sharply different karyotypes and undoubtedly belong to different species. In this context, the taxonomic status and the relationship between the two chromosomal forms in Sinai (2 n  = 36) and Israel (2 n  = 38), chromosomally homozygous across a vast range except for a very narrow hybrid zone, remain poorly documented. Neither of these forms have previously been studied by chromosome banding; thus, the exact nature of chromosomal differences as well as the species to which these forms should be assigned remain unknown. Here, we present the data on comparative G-banding analysis and morphometrics of Acomys from Israel, Sinai, and Saudi Arabia, and a hybrid obtained in laboratory crosses between latter two. The analysis revealed that karyotype of Acomys from Israel is identical to that of Acomys from Saudi Arabia and both are different from that of Acomys from Sinai by one Robertsonian fusion. Therefore, karyotypically, all three are very different from A. cahirinus . It follows from the study that Sinai and probably Arabian peninsula and Minor Asia must be excluded from geographical distribution of A. cahirinus , which is limited from West Sahara to Egypt along Nile river (except Sinai). Furthermore, the synthesis of chromosomal and recent molecular data suggests a phylogeographical scenario explaining the modern distribution of Acomys in the Sinai and Arabian peninsulas and permits the update of the taxonomic status of these populations.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 203–214.     

Phylogeography of the South China field mouse (Apodemus draco) on the southeastern Tibetan Plateau reveals high genetic diversity and glacial refugia

The southeastern margin of the Tibetan Plateau (SEMTP) is a particularly interesting region due to its topographic complexity and unique geologic history, but phylogeographic studies that focus on this region are rare. In this study, we investigated the phylogeography of the South China field mouse, Apodemus draco, in order to assess the role of geologic and climatic events on the Tibetan Plateau in shaping its genetic structure. We sequenced mitochondrial cytochrome b (cyt b) sequences in 103 individuals from 47 sampling sites. In addition, 23 cyt b sequences were collected from GenBank for analyses. Phylogenetic, demographic and landscape genetic methods were conducted. Seventy-six cyt b haplotypes were found and the genetic diversity was extremely high (π = 0.0368; h = 0.989). Five major evolutionary clades, based on geographic locations, were identified. Demographic analyses implied subclade 1A and subclade 1B experienced population expansions at about 0.052-0.013 Mya and 0.014-0.004 Mya, respectively. The divergence time analysis showed that the split between clade 1 and clade 2 occurred 0.26 Mya, which fell into the extensive glacial period (EGP, 0.5-0.17 Mya). The divergence times of other main clades (2.20-0.55 Mya) were congruent with the periods of the Qingzang Movement (3.6-1.7 Mya) and the Kun-Huang Movement (1.2-0.6 Mya), which were known as the most intense uplift events in the Tibetan Plateau. Our study supported the hypothesis that the SEMTP was a large late Pleistocene refugium, and further inferred that the Gongga Mountain Region and Hongya County were glacial refugia for A. draco in clade 1. We hypothesize that the evolutionary history of A. draco in the SEMTP primarily occurred in two stages. First, an initial divergence would have been shaped by uplift events of the Tibetan Plateau. Then, major glaciations in the Pleistocene added complexity to its demographic history and genetic structure.     

Preliminary genetic characterization of two lineages of black rats (Rattus rattus sensu lato) in Japan, with evidence for introgression at several localities

We conducted a pilot survey of genetic diversity among 37 karyotyped individuals of the black rat Rattus rattus (sensu lato) from six localities on the Japanese Islands, using complete gene sequences of mitochondrial cytochrome b (cyt b) and nuclear interphotoreceptor retinoid binding protein (IRBP). Our sampling included two previously documented karyotypic groups: 'Oceanian' with 2n = 38 and 'Asian' with 2n = 42. Cyt b sequences for most individuals clustered according to their karyotypic groups, with an average between-group divergence of 3.8%. One exception was that individuals from Kagoshima (Kyushu Island) showed 'Asian' karyotypes combined with a cyt b haplotype that differed by a single nucleotide substitution from the haplotype of the 'Oceanian' karyotypic group. Six IRBP haplotypes were identified. They belonged to three distinct IRBP lineages (I-III), with an average inter-lineage divergence of 1%. Among homozygous individuals, these lineages showed good association with the karyotypic groups: IRBP lineage I occurred only with 'Oceanian' karyotypes, while IRBP lineages II and III both occurred with 'Asian' karyotypes. Individuals from Kagoshima all possessed IRBP of 'Asian' lineages, despite the presence of an 'Oceanian' mitochondrial type. The Chichijima population (Ogasawara Islands) featured exclusively 'Asian' karyotypes and cyt b sequences, but various combinations of all three IRBP lineages. The Kagoshima and Chichijima populations thus provide strong evidence of viable hybridization and genetic introgression between the two karyotypic groups, but with variable genetic outcomes. Our results demonstrate the potential of combined analysis of karyotypes and mitochondrial and nuclear gene sequences to elucidate the complex dispersal and population history of the black rat.     

How many species of Apodemus and Rattus occur in China? A survey based on mitochondrial cyt b and morphological analyses

Apodemus(mice) and Rattus(rats) are the top rodent reservoirs for zoonoses in China, yet little is known about their diversity. We reexamined the alpha diversity of these two genera based on a new collection of specimens from China and their cyt b sequences in Gen Bank. We also tested whether species could be identified using external and craniodental measurements exclusively. Measurements from 147 specimens of Apodemus and 233 specimens of Rattus were used for morphological comparisons. We analysed 74 cyt b sequences of Apodemus and 100 cyt b sequences of Rattus to facilitate phylogenetic estimations. Results demonstrated that nine species of Apodemus and seven species of Rattus, plus a new subspecies of Rattus nitidus, are distributed in China. Principal component analysis using external and craniodental measurements revealed that measurements alone could not separate the recognized species. The occurrence of Rattus pyctoris in China remains uncertain.     

Molecular phylogeny of avian genus Syrmaticus based on the mitochondrial cytochrome B gene and control region

Mitochondrial DNA cytochrome b (cyt b) and control region (CR) nucleotide sequences were used to study the molecular phylogeny of the genus Syrmaticus. We found that the substitution rates among the three codon positions of cyt b were heterogeneous and the transition-transversion ratio was highly biased. As to CR sequences of the genus, most variable sites were in the peripheral domains. All molecular phylogenetic trees based on the two genes showed that: 1) the Syrmaticus was monophyletic and included five species with the following cladistic relationship: (S. reevesii, (S. soemmerringii, (S. mikado, (S. humiae and S. ellioti)))). Using the TN genetic distance of cyt b, we inferred the divergence time of the five species according to putative molecular clock and found that values were largely in agreement with the geological scenarios. The origin and speciation processes of the studied group were inferred by combining molecular and biogeographical evidences.     

Molecular phylogenetic analysis of circumnuclear hemoproteids (Haemosporida: Haemoproteidae) of sylviid birds, with a description of Haemoproteus parabelopolskyi sp. nov

Haemoproteus spp., with circumnuclear gametocytes and tentatively belonging to Haemoproteus belopolskyi, are widespread and prevalent in warblers belonging to the Sylviidae, with numerous mitochondrial cytochrome b (cyt b) lineages detected among them.We sampled the hemoproteids from 6 species of warblers adjacent to the Baltic Sea. Parasites were identified to species based on morphology of their gametocytes, and a segment of the parasite's cyt b gene was sequenced. Sixteen mitochondrial cyt b lineages of hemoproteids with circumnuclear gametocytes were recorded. Two clades of lineages (clade A in species of Acrocephalus and Hippolais and clade B in species of Sylvia) with sequence divergence between their lineages >5% are distinguished in the phylogenetic tree. Within the clades A and B, the genetic distance between the lineages is < or = 3.9 and < and = 2.8%, respectively. We compared the morphology of gametocytes of 3 lineages (hHIICT1, hMW1, and hSYAT2) in detail. The lineages hHIICTI and hMW1 (clade A) belong to the morphospecies H. belopolskyi. Parasites of the lineage hSYAT2 (clade B) are described as a new species Haemoproteus parabelopolskyi, which can be readily distinguished from H. belopolskyi by the significantly smaller nuclei of its macrogametocytes. Lineages closely related to H. belopolskyi and H. parabelopolskyi are identified. The sequence divergence between lineages of these 2 morphospecies ranges between 5.3 and 8.1%. It seems probable that avian Haemoproteus spp. with a genetic differentiation of > or =5% in mitochondrial cyt b gene might be morphologically differentiated at the stage of gametocytes. This study establishes the value of both PCR and morphology in identification of avian hemoproteids.     

Mitochondrial DNA phylogeny and morphological diversity in the genus Mastus (Beck, 1837): a study in a recent (Holocene) island group (Koufonisi, south-east Crete)

Three endemic Cretan land snail species of the genus Mastus (Beck, 1837) from the island group of Koufonisi (south-east Crete) and the eastern part of Crete, were studied by multivariate analysis of shell morphology and analysis of mtDNA sequences. The phylogeny of the populations studied and the processes effecting the genetic and morphological diversity of the species were investigated. Extremely high mtDNA sequence divergence was observed both within and between populations. The Cretan populations could not be distinguished morphologically, while the populations of the islets were more distinct. We argue that the active geological past of the area (including sea level changes) and the long-term presence of humans has produced a mixing up of Mastus populations leading to the accumulation of high divergence of mtDNA sequences on a small spatial scale. The limited morphological diversity and the distinct shell 'identity' of the islets' populations can be attributed to the selective pressures of the island group.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 78, 383–399.     

A mitogenome of the Chevrier's field mouse (Apodemus chevrieri) and genetic variations inferred from the cytochrome b gene

The Chevrier's field mouse (Apodemus chevrieri) is an endemic species to China and is an important pest in agriculture and human diseases. In this study, the complete mitochondrial genome of this species was sequenced and its size was 16,298 bases (accession no.: HQ896683). The mitogenome structure was similar compared with other reported rodent mitochondrial genomes and includes 13 protein-coding genes, 2 rRNA genes (12S rRNA and 16S rRNA), 22 tRNA genes, and 1 control region. This was the first complete mitogenome sequenced in genus Apodemus. The phylogenetic analyses based on the sequences of 12 heavy-strand protein-coding genes demonstrated that A. chevrieri clustered together with genus Mus. Additionally, extremely high haplotype and nucleotide diversities (h=0.978, π=2.6%) were observed based on 44 mitochondrial cytochrome b (cyt b) gene sequences. This suggests adaptive divergence of this species to a variety of living habitats and potential refuges in the eastern margin of the Hengduan Mountains during the Quaternary ice ages. No population expansions or genetic bottlenecks were observed in demographic analyses. The phylogenetic analysis of cyt b sequences and haplotypes revealed a genetic differentiation between north and south populations. The divergence between north clade and south clade occurred probably in the middle Pleistocene 1.1815 million years ago (Mya) (95% highest posterior density 2.3189-0.2737 Mya), which was congruent with the periods of the most tense uplift events in the Tibetan Plateau.     

B chromosome ancestry revealed by histone genes in the migratory locust

In addition to the standard set of chromosomes (A), about 15% of eukaryote genomes carry B chromosomes. In most cases, B chromosomes behave as genomic parasites being detrimental for the individuals carrying them and prospering in natural populations because of transmission advantages (drive). B chromosomes are mostly made up of repetitive DNA sequences, especially ribosomal DNA (rDNA), satellite DNA and mobile elements. In only two cases have B chromosomes been shown to carry protein-coding genes. Although some B chromosomes seem to have derived from interspecific hybridisation, the most likely source of B chromosomes is the host genome itself, but the specific A chromosome being the B ancestor has not been identified in any B-containing species. Here, we provide strong evidence for B chromosome ancestry in the migratory locust, based on the location of genes for the H3 and H4 histones in the B chromosome and a single A chromosome pair (i.e. the eighth in order of decreasing size). The high DNA sequence similarity of A and B chromosome H3–H4 genes supports B-origin from chromosome 8. The higher variation shown by B sequences, compared to A sequences, suggests that B chromosome sequences are most likely inactive and thus less subjected to purifying selection. Estimates of time of divergence for histone genes from A and B chromosomes suggest that B chromosomes are quite old (>750,000 years), showing the B-chromosome ability to persist in natural populations for long periods of time.     

Low mitochondrial DNA variation among American alligators and a novel non-coding region in crocodilians

We analyzed 1317-1823 base pairs (bp) of mitochondrial DNA sequence beginning in the 5' end of cytochrome b (cyt b) and ending in the central domain of the control region for 25 American alligators (Alligator mississippiensis) and compared these to a homologous sequence from a Chinese alligator (A. sinensis). Both species share a non-coding spacer between cyt b and tRNA(Thr). Chinese alligator cyt b differs from that of the American alligator by 17.5% at the nucleotide level and 13.8% for inferred amino acids, which is consistent with their presumed ancient divergence. Only two cyt b haplotypes were detected among the 25 American alligators (693-1199 bp surveyed), with one haplotype shared among 24 individuals. One alligator from Mississippi differed from all other alligators by a single silent substitution. The control region contained only slightly more variation among the 25 American alligators, with two variable positions (624 bp surveyed), yielding three haplotypes with 22, two, and one individuals in each of these groups. Previous genetic studies examining allozymes and the proportion of variable microsatellite DNA loci also found low levels of genetic diversity in American alligators. However, in contrast with allozymes, microsatellites, and morphology, the mtDNA data shows no evidence of differentiation among populations from the extremes of the species range. These results suggest that American alligators underwent a severe population bottleneck in the late Pleistocene, resulting in nearly homogenous mtDNA among all American alligators today.     

Phylogeographic Pattern and Extensive Mitochondrial DNA Divergence Disclose a Species Complex within the Chagas Disease Vector Triatoma dimidiata

Background

Triatoma dimidiata is among the main vectors of Chagas disease in Latin America. However, and despite important advances, there is no consensus about the taxonomic status of phenotypically divergent T. dimidiata populations, which in most recent papers are regarded as subspecies.

Methodology and Findings

A total of 126 cyt b sequences (621 bp long) were produced for specimens from across the species range. Forty-seven selected specimens representing the main cyt b clades observed (after a preliminary phylogenetic analysis) were also sequenced for an ND4 fragment (554 bp long) and concatenated with their respective cyt b sequences to produce a combined data set totalling 1175 bp/individual. Bayesian and Maximum-Likelihood phylogenetic analyses of both data sets (cyt b, and cyt b+ND4) disclosed four strongly divergent (all pairwise Kimura 2-parameter distances >0.08), monophyletic groups: Group I occurs from Southern Mexico through Central America into Colombia, with Ecuadorian specimens resembling Nicaraguan material; Group II includes samples from Western-Southwestern Mexico; Group III comprises specimens from the Yucatán peninsula; and Group IV consists of sylvatic samples from Belize. The closely-related, yet formally recognized species T. hegneri from the island of Cozumel falls within the divergence range of the T. dimidiata populations studied.

Conclusions

We propose that Groups I–IV, as well as T. hegneri, should be regarded as separate species. In the Petén of Guatemala, representatives of Groups I, II, and III occur in sympatry; the absence of haplotypes with intermediate genetic distances, as shown by multimodal mismatch distribution plots, clearly indicates that reproductive barriers actively promote within-group cohesion. Some sylvatic specimens from Belize belong to a different species – likely the basal lineage of the T. dimidiata complex, originated ∼8.25 Mya. The evidence presented here strongly supports the proposition that T. dimidiata is a complex of five cryptic species (Groups I–IV plus T. hegneri) that play different roles as vectors of Chagas disease in the region.     

Multiple cryptic species of sympatric generalists within the avian blood parasite Haemoproteus majoris

The avian haemosporidian parasite Haemoproteus majoris has been reported to infect a wide range of passerine birds throughout the Holarctic ecozone. Five cytochrome b (cyt b) lineages have been described as belonging to the morphological species H. majoris, and these form a tight phylogenetic cluster together with 13 undescribed lineages that differ from each other by < 1.2% in sequence divergence. Records in a database (MalAvi) that contains global findings of haemosporidian lineages generated by universal primers suggest that these lineages vary substantially in host distribution. We confirm this pattern in a data set collected at Lake Kvismaren, Sweden, where three of the generalist lineages have local transmission. However, whether these lineages represent intraspecific mitochondrial diversity or clusters of cryptic species has previously not been examined. In this study, we developed novel molecular markers to amplify the partial segments of four nuclear genes to determine the level of genetic diversity and gene phylogenies among the five morphologically described cyt b lineages of H. majoris. All five cyt b lineages were strongly associated with unique nuclear alleles at all four nuclear loci, indicating that each mitochondrial lineage represents a distinct biological species. Within lineages, there was no apparent association between nuclear alleles and host species, indicating that they form genetically unstructured populations across multiple host species.     

Molecular phylogeny of Japanese Leporidae,the Amami rabbit Pentalagus furnessi,the Japanese hare Lepus brachyurus,and the mountain hare Lepus timidus,inferred from mitochondrial DNA sequences

We determined mitochondrial 12S ribosomal RNA (rRNA) and cytochrome b (cyt b) gene sequences in three leporid species of Japan, the Amami rabbit Pentalagus furnessi from the Ryukyu Islands, the Japanese hare Lepus brachyurus from Honshu, and a Japanese form of the mountain hare Lepus timidus ainu from Hokkaido. We compared the sequences with those of other taxa of leporids available in databases. Phylogenetic trees of the 12S rRNA gene sequences indicated that the lineage of P. furnessi diversified during the generic radiation of the leporids at an ancient time, which was estimated to have been the middle Miocene. Cyt-b gene trees revealed that the lineage of L. brachyurus branched off at an early stage in the speciation of Lepus, probably at the beginning of the Pliocene. The cyt b sequences of L. t. ainu were somewhat distinct from those of continental conspecific populations; this lineage divergence is likely to have occurred during the middle or late Pleistocene. The results show that the three regions of the Japanese archipelago, Ryukyu, Honshu-Shikoku-Kyushu, and Hokkaido, now preserve their own leporid taxa, each with a different extent of genetic endemicity. It is possible that the zoogeographic traits of the Japanese leporids are a consequence of the evolutionary dynamics of leporids in East Asia, in that the radiation centers of leporids are likely to have shifted from tropical, through temperate, to arctic zones.     

Species identification in the taxonomically neglected, highly diverse, neotropical parasitoid wasp genus Notiospathius (Braconidae: Doryctinae) based on an integrative molecular and morphological approach

Various DNA sequence-based methods for species delineation have recently been developed to assess the species-richness of highly diverse, neglected invertebrate taxa. These methods, however, need to be tested under a variety of conditions, including the use of different markers and parameters. Here, we explored the species diversity of a species-rich group of braconid parasitoid wasps, the Neotropical genus Notiospathius, including 233 specimens from 10 different countries. We examined sequences of two mitochondrial (mt) (COI, cyt b) and one nuclear (wg) gene fragments. We analysed them separately as well as concatenating the mt data with the general mixed Yule-coalescent (GMYC) model for species delineation using different tree-building methods and parameters for reconstructing ultrametric trees. We evaluated the performance of GMYC analyses by comparing their species delineations with our morphospecies identifications. Reconstructing ultrametric trees with a relaxed lognormal clock rate using the program BEAST gave the most congruent results with morphology for the two mt markers. A tree obtained with wg using the programs MrBayes+Pathd8 had the fewest cases of incongruence with morphology, though the performance of this nuclear marker was considerably lower than that of COI and cyt b. Species delimitation using the coalescent prior to obtain ultrametric trees was morphologically more congruent with COI, whereas the Yule prior was more congruent with cyt b. The analyses concatenating the mt datasets failed to recover some species supported both by morphology and the separate analyses of the mt markers. The highest morphological congruence was obtained with the GMYC analysis on an ultrametric tree reconstructed with cyt b using the relaxed lognormal clock rate and the Yule prior, thus supporting the importance of using alternative markers when the information of the barcoding locus (COI) is not concordant with morphological evidence. Seventy-one species were delimited based on the congruence found among COI, cyt b and morphology. Both mt markers also revealed the existence of seven potential cryptic species. This high species richness from a scattered geographical sampling indicates that there is a remarkable number of Notiospathius species that remains undiscovered.     

蜂猴线粒体细胞色素b基因变异特点及系统发育分析

测定了蜂猴属（Nycticebus)1个蜂猴（N.coucang)和2个矮蜂猴（N,pygmaseus)个体的线粒体细胞色素b(cyt-b)基因全序列,比较现有的司猴科其他种序列,分析了核苷酸序列差异和碱基替换特点,以指猴为外群重建了系统发育树,结果表明,在所研究的个体中,2个蜂猴物种碱基组成具有哺乳动物的共同特点,它们之间转换比（特别是密码子第3位）是颠换比的6倍多,大于其他种间比较;低的Ka/Ks值（＜0．1）,说明懒猴科cyt-b基因的异义突位点受到强的选择压力作用。由cyt-b基因构建的系统发育树符合懒猴科化石记录和形态学分类观点,根据化石记录和与分化时间有一定线性关系的第3位颠换和同义突变速率,估算蜂猴与倭蜂猴种间,蜂猴与蜂属间可能的分化时间分别为300和600万年。     

Evolution and function of B chromosome 45S rDNA sequences in Brachycome dichromosomatica.

The origin and activity of 45S rDNA located on micro B chromosomes of the daisy Brachycome dichromosomatica were analysed. The internal transcribed spacer 2 (ITS2) of the 45S rRNA gene was sequenced for micro B, large B, and A chromosomes of B. dichromosomatica cytodeme A2, and conserved differences were identified between sequences originating from A and both types of B chromosomes. Phylogenetic analysis did not identify a species containing an ITS2 sequence more similar to either of the B chromosome sequences than the B. dichromosomatica A chromosome sequences. Thus, an origin of the B chromosomes from A chromosomes at a time prior to the divergence of the 4 cytodemes of B. dichromosomatica is suggested. The frequent (70%) nucleolar non-association of micro B chromosomes suggests inactivity of micro B 45S rDNA.