Molecular Phylogeny of the Lardizabalaceae Based on TrnL-F Sequences and Combined Chloroplast Data

The molecular phylogeny of the Lardizabalaceae is reconstructed based on chloroplast trn L_F sequences alone and combined trn L_F and rbc L sequences. The phylogenetic topologies agree well with Qin's and Takhtajan's tribal classification in both analyses. Decaisneae and Sinofranchetieae are basal clades in the phylogenetic trees and external to all other taxa in the family. Lardizabaleae consisting of Boquila and Lardizabala are well supported in both trn L_F (100%) analysis and trn L_F and rbc L combined analysis (99%). Tribe Akebieae are strongly supported by a bootstrap value of 100% in both trn L_F analysis and trn L_F and rbc L combined analysis. However, the new genus Archakebia is nested within the genus Akebia in the trn L_F trees. In the combined trees, Archakebia is sister to Akebia with high bootstrap support. The inter_relationships among three closely related genera Parvatia , Holboellia and Stauntonia are still problematic. P. brunoniana ssp. elliptica is sister to H. latifolia in both analyses with low bootstrap support. H. parviflora is nested within the Stauntonia and sister to S. cavalerieana . Therefore, these three genera of tribe Akebieae may not be monophylytic and their generic boundary and delimitation need to be further studied, by exploring more molecular data, together with more morphological characters.

Molecular Phylogeny of Bisexual Artemia Based on 16S rDNA

[Objective] The research aimed at investigating the taxonomic position and phylogenetic relationship of bisexual brine shrimps.[Method] 16S rDNA of three species of bisexual Artemia from China was determined;the homologous sequences between them and 11 relative species of Artemia from GenBank were compared;the molecular phylogenetic tree was constructed by Mega Microsoft using Artemiopsis stefanssoni as outgroup.[Result] Artemia persimilis was the primal group in genus Artemia;Artemia franciscana and Artemia monica were the evolved groups;Artemia urmiana,Artemia sinica and other Artemia species from China shared a close genetic relationship.[Conclusion] Based on the 16S rDNA sequence of them,the phylogenetic relationships of these bisexual Artemia species were A.persimilis→A.urmiana,A.sinica and A.tibetiana→A.tunisiana→A.monica→A.Franciscan.

Molecular Phylogeny of Slow Lorises (Nycticebus) Revealed by D-loop Sequences and Complete Cytochrome b Gene Sequences of Mitochondrial DNA

Partial sequences of the D-loop and the complete sequences of cytochrome b gene (1 140 bp) of the slow lorises (genus Nycticebus) were undertaken to investigate evolutionary relationships among species of Nycticebus.Sequence analysis results consistently provide new taxonomy evidence at the DNA level for supporting Ratajszczak and Groves’ viewpoint that N.intermedus is merely the adult of N.pygmaeus (Ratajszczak,1998;Groves,1971).Phylogenetic analysis was performed by means of the combined data and these two separate sequences data,respectively,by using various methods,supporting the same topology,in which genus Nycticebus was formed of two clusters.The first cluster was composed of N.pygmaeus,and the second cluster of N.coucang.It also could provide a new molecular genetic evidence to support the view that the genus comprises two species:N.coucang and N.pygmaeus.

Morphology and molecular phylogeny of Paragorgia rubra sp.nov.(Cnidaria:Octocorallia),a new bubblegum coral species from a seamount in the tropical Western Pacific

A new species of bubblegum coral, Paragorgia rubra sp. nov., discovered from a seamount at a water depth of 373 m near the Yap Trench is studied using morphological and molecular approaches. Paragorgia rubra sp. nov. is the fourth species of the genus found in the tropical Western Pacific. The new gorgonian is red-colored, uniplanar, and measures approximately 530 mm high and 440 mm wide, with autozooids distributed only on one side of the colony. Paragorgia rubra sp. nov. is most similar to P. kaupeka Sainchez, 2005, but differs distinctly in the polyp ovals with large and compound protuberances （vs. small and simple conical protuberances） and the medullar spindles possessing simple conical protuberances （vs. compound protuberances）. Moreover, P. rubra sp. nov. differs from P. kaupeka in the smaller length/ width ratio of surface radiates （1.53 vs. 1.75 ）. The genetic distance of the mtMutS gene between P. rubra sp. nov. and P. kaupeka is 0.66%, while the intraspecific distances within Paragorgia Milne-Edwards ＆ Haime, 1857 except the species P. regalis complex are no more than 0.5%, further supporting the establishment of the new species. Furthermore, the ITS2 secondary structure ofP. rubra sp. nov. is also different from those of congeners. Phylogenetic analyses indicate Paragorgia rubra sp. nov. and P. kaupeka form a clade, which branched early within Paragorgia and diversified approximately 15 Mya.

Molecular phylogeny,biogeography and character evolution of the montane genus Incarvillea Juss.(Bignoniaceae)

The complex orogeny of the Himalaya and the Qinghai-Tibet Plateau(QTP)fosters habitat fragmentation that drives morphological differentiation of mountain plant species.Consequently,determining phylogenetic relationships between plant subgenera using morphological characters is unreliable.Therefore,we used both molecular phylogeny and historical biogeographic analysis to infer the ancestral states of several vegetative and reproductive characters of the montane genus Incarvillea.We determined the taxonomic position of the genus Incarvillea within its family and inferred the biogeographical origin of taxa through Bayesian inference(BI),maximum likelihood(ML)and maximum parsimony(MP)analyses using three molecular data sets(trnL-trnF sequences,nr ITS sequences,and a data set of combined sequences)derived from 81%of the total species of the genus Incarvillea.Within the genus-level phylogenetic framework,we examined the character evolution of 10 key morphological characters,and inferred the ancestral area and biogeographical history of the genus.Our analyses revealed that the genus Incarvillea is monophyletic and originated in Central Asia during mid-Oligocene ca.29.42 Ma.The earliest diverging lineages were subsequently split into theWestern Himalaya and Sino-Himalaya during the early Miocene ca.21.12 Ma.These lineages resulted in five re-circumscribed subgenera(Amphicome,Olgaea,Niedzwedzkia,Incarvillea,and Pteroscleris).Moreover,character mapping revealed the ancestral character states of the genus Incarvillea(e.g.,suffruticose habit,cylindrical capsule shape,subligneous capsule texture,absence of capsule wing,and loculicidal capsule dehiscence)that are retained at the earliest diverging ancestral nodes across the genus.Our phylogenetic tree of the genus Incarvillea differs from previously proposed phylogenies,thereby recommending the placement of the subgenus Niedzwedzkia close to the subgenus Incarvillea and maintaining two main divergent lineages.

Genetic Diversity of MHC Gene and Molecular Phylogeny in Wanbei Cattle

[Objective] To investigate the genetic diversity, molecular phylogeny and origination in Wanbei cattle. [Methedl Blood samples were collected from 50 Wanbei cattle and 14 local cattle breeds. According to the bovine MHC gene sequence, one pair of primers was designed and used for PCR amplification. The sequences of amplified MHC genes were aligned to detect MHC haplotypes and nucleotide polyrnorphisms. A phy- Iogenetic tree based on the MHC gene was also constructed. [Result] A total of 24 polymorphic loci covering 7.86% of the entire length of the se- quences and seven MHC haplotypes were detected. Among these polymorphic loci, there were 18 transitions, 1 tranversions and 5 transition / tran- version. The difference in nucleotide sequence was 0.26% -4.53% between the haplotypes. The nucleotide diversity （π value） and haplotype di- versity （H） were 0.012 - 0.019 and 0.602 - 0.617, respectively. [ Conclusion ] Wanbei cattle do not have rich MHC genetic diversity. They have closer aenetic relationship with Luxi cattle, and both are within the same sister group.

Molecular phylogeny of Bactrocera species （Diptera： Tephritidae） inferred from mitochondrial 16s rDNA sequences

The fruit flies of the Bactrocera （Diptera： Tephritidae） are one of the major economically important insects in Asia. So far, there are at least seven species found in Chongqing, China. By using mitochondrial 16s rDNA sequences, the phylogenetic relationships among seven Bactrocera species, Bactrocera cucurbitae （Coquillett）, B. tau （Walker）, B. diaphora （Hendel）, B. caudata （Fabricius）, B. scutellata （Hendel）, B. dorsalis （Hendel）, and B. minax （Enderlein） were investigated. Nucleotide diversity within species ranged from 0.3% to 10.9%. According to the result, B. cucurbitae （Coquillett）, B. tau （Walker）, B. diaphora （Hendel）, and B. caudata （Fabricius） have no enough sites to be distinguished among the other species in the 347bp nucleotide sequences of the 16s rDNA gene. At the same time, excluding B. scutellata （Hendel） and B. minax （Enderlein）, the other species recognition sites are not only too little but also discontinuous. Phylogenetic trees calculated from both maximum parsimony and neighbor-joining phylogenetic analysis methods showed that B. cucurbitae and B. tau were closely related, B. diaphora （Hendel） and B. caudata （Fabricius） also had a close relationship. B. minax （Enderlein） has a furthest relationship from the other 6 species. From the results, we can infer that the our data supports previous classification of Bactrocera based on morphological characters in other words, but the Bactrocera subgenera, Zeugodacus is polyphyletic.

Phylogeny and Geographical Derivation of Genus Hildenbrandia Based on Morphological and Molecular Evidence

The marine and freshwater Hildenbrandia samples were collected from diverse locations of China and Japan,and mor-phological characterization and molecular phylogenetic analysis were conducted on these samples.Morphological measurements of freshwater specimens were consistent with the results of H.jigongshanensis,whereas cell dimensions of marine specimens were slightly larger than those of widely distributed H.rubra and H.crouanii.Phylogenetic trees based on rbcL,psbA and UPA sequences were consistent.Freshwater specimens collected in this study formed an independent clade with H.jigongshanensis supported by high values.Species attribution was confirmed further by the similar ITS2 secondary structures among the samples of H.jigongshan-ensis.It was found that the intraspecific divergence of rbcL gene in H.jigongshanensis was smaller than other two freshwater species H.rivularis and H.angolensis.Phylogenetic trees showed that marine specimens in this study grouped together with H.rubra samples of North America,and were different with the H.rubra samples of Europe.Combining the comparison results of CBC numbers in ITS2 secondary structure,we propose the marine samples collected in this study to be a new species Hildenbrandia qingdaoensis sp.nov.The separation of the marine and freshwater specimens was supported by the rbcL,UPA and psbA phylogeny as well as ITS2 se-condary structure inference.Biogeographical reconstruction showed the ancestor of freshwater Hildenbrandia might derive from South America and dispersed from North America to Europe and then to Asian countries,which needs to be verified further with more sam-ples and molecular evidences from South America.

Morphological and Molecular Data Revealed One New Species of the Short-legged Toads Brachytarsophrys Tian and Hu,1983(Anura,Megophryidae)from Yunnan,China

A new species of the genus Brachytarsophrys,named Brachytarsophrys wenshanensis sp.nov.,has been identified in southeastern Yunnan,China.This new species can be readily distinguished from other known congeners by both morphological criterion and molecular analysis of three mitochondrial gene segments:16S,COI,and Cytb.This classification is based on the following morphological characters:(1)medium body size(SVL 83.8–85.1 mm in two adult males);(2)enormous head,with head width nearly 1.2 times the length;(3)tongue pyriform,feebly notched;(4)non-meeting heels;(5)male lacking nuptial pad;(6)tibiotarsal articulation reaching angle of mouth when hindlimbs are extended forward alongside the body;(7)absence of outer metatarsal tubercle,inner metatarsal tubercle elliptic and approximately equal to first toe;(8)rudimentary toe webbing,webbing formula:Ⅰ(2–)–(2^(++))Ⅱ(2^(–))–(3^(++))Ⅲ(2^(½))–(4)Ⅳ(4^(+))–(2^(⅔))V;(9)lateral fringes narrow;(10)dermal ridge or glandular fold on dorsum absent;(11)pectoral glands distinct and irregular,femoral gland small.Our work increases the number of species within the genus Brachytarsophrys to 9.

DNA barcoding of genus Toxoptera Koch （Hemiptera： Aphididae） Identification and molecular phylogeny inferred from mitochondrial COl sequences

Identification of aphid species is always difficult due to the shortage of easily distinguishable morphological characters. Aphid genus Toxoptera consists of species with similar morphology and similar to Aphis in most morphological characters except the stridulatory apparatus. DNA barcodes with 1 145 bp sequences of partial mitoehondrial cytochrome-coxidase I （COI） genes were used for accurate identification of Toxoptera. Results indicated mean intraspecific sequence divergences were 1.33%, whereas mean interspecific divergences were greater at 8.29% （0.13% and 7.79% if T. aurantii 3 and T. aurantii 4 are cryptic species）. Sixteen samples were distinguished to four species correctly by COI barcodes, which implied that DNA barcoding was successful in discrimination of aphid species with similar morphology. Phylogenetic relationships among species of this genus were tested based on this portion of COI sequences. Four species of Toxoptera assembled a clade with low support in maximum-parsimony （MP） analysis, maximumlikelihood （ML） analysis and Bayesian phylogenetic trees, the genus Toxoptera was not monophyletic, and there were two sister groups, such as T. citricidus and T. victoriae, and two clades of T. aurantii which probably presented cryptic species in the genus.

Molecular phylogeny of Pamphagidae （Acridoidea, Orthoptera） from China based on mitochondrial cytochrome oxidase II sequences

Phylogenetic relationships of Pamphagidae were examined using cytochrome oxidase subunit II （COIl） mtDNA sequences （684 bp）. Twenty-seven species of Acridoidea from 20 genera were sequenced to obtain mtDNA data, along with four species from the GenBank nucleotide database. The purpose of this study was analyzing the phylogenetic relationships among subfamilies within Pamphagidae and interpreting the phylogenetic position of this family within the Acridoidea superfamily. Phylogenetic trees were reconstructed using neighbor-joining （NJ）, maximurn parsimony （MP） and Bayesian inference （BI） methods. The 684 bp analyzed fragment included 126 parsimony informative sites. Sequences diverged 1.0%-11.1% between genera within subfamilies, and 8.8%-12.3% between subfamilies. Amino acid sequence diverged 0-6.1% between genera within subfamilies, and 0.4%-7.5% between subfamilies. Our phylogenetic trees revealed the monophyly of Pamphagidae and three distinct major groups within this family. Moreover, several well supported and stable clades were found in Pamphagidae. The global clustering results were similar to that obtained through classical morphological classification： Prionotropisinae, Thrinchinae and Pamphaginae were monophyletic groups. However, the current genus Filchnerella （Prionotropisinae） was not a monophyletic group and the genus Asiotmethis （Prionotropisinae） was a sister group of the genus Thrinchus （Thrinchinae）. Further molec- ular and morphological studies are required to clarify the phylogenetic relationships of the genera Filchnerella and Asiotmethis.

Phylogeny of Apaturinae Butterflies (Lepidoptera: Nymphalidae) Based on Mitochondrial Cytochrome OxidaseⅠ Gene

The phylogenetic relationships of genera in the subfamily Apaturinae were examined using mtDNA sequence data from 1,471 bp of cytochrome oxidase subunit Ⅰ （COI）. The mitochondrial COI gene from a total of 16 species in 11 genera were sequenced to obtain mtDNA data, along with those of 4 species obtained from GenBank, to construct the MP and the NJ trees using Athyma jina, Penthema adelma, Polyura nepenthes, and Charaxes bernardus as outgroups. The transitions at the third codon positions of the COI data set were found saturated, but they were retained for analysis, because they contain the majority of the phylogenetic information. The impacts of equal weight assumptions for all characters in the parsimonious analysis were assessed by potential alternations in clades in response to different transition/transversion weighting schemes. The results indicated four distinct major groups in Apaturinae. Moreover, several well supported and stable clades were found in the Apaturinae. The study also identified undetermined taxon groups whose positions were weakly supported and were subject to changes under different weighting schemes. Within the Apaturinae, the clustering results are approximately identical to the classical morphological classification. The mtDNA data suggest the genus Mimathyma as a monophyletic group. Lelecella limenitoides and Dilipa fenestra have close relationship with very strong support in all phylogenetic trees. It also supports the taxonomic revision of removing several species from Apatura to other genera, namely Mimathyma schrenckii, M. chevana, M. nycteis, Chitoria subcaerulea, C. fasciola, C. pallas, and Helcyra subalba.

Identification, pathogenicity, and fungicide sensitivity of Eutiarosporella dactylidis associated with leaf blight on maize in China

Maize(Zea mays L.) is an economically vital grain crop that is cultivated worldwide. In 2011, a maize foliar disease was detected in Lingtai and Lintao counties in Gansu Province, China. The characteristic signs and symptoms of this disease include irregular chlorotic lesions on the tips and edges of infected leaves and black punctate fruiting bodies in dead leaf tissues. Given favourable environmental conditions, this disease spread to areas surrounding Gansu. In this study, infected leaves were collected from Gansu and Ningxia Hui Autonomous Region between 2018and 2020 to identify the disease-causing pathogen. Based on morphological features, pathogenicity tests, and multilocus phylogenetic analysis involving internal transcribed spacer(ITS), 18S small subunit rDNA(SSU), 28S large subunit rDNA(LSU), translation elongation factor 1-alpha(TEF), and β-tubulin(TUB) sequences, Eutiarosporella dactylidis was identified as the causative pathogen of this newly discovered leaf blight. Furthermore, an in vitro bioassay was conducted on representative strains using six fungicides, and both fludioxonil and carbendazim were found to significantly inhibit the mycelial growth of E. dactylidis. The results of this study provide a reference for the detection and management of Eutiarosporella leaf blight.

Taxonomy of Two New Peritrich Ciliates(Protozoa:Ciliophora)from Inland Saltwaters of China

The inland saltwater lakes harbor exceptional biodiversity.Here,two new species of solitary sessile peritrich ciliates were isolated from Qinghai Lake,the largest inland saltwater lake in China.Their morphology,ciliature,silverline system,and molecular phylogeny were investigated based on live observation,silver staining,and analysis of the small subunit ribosomal DNA(SSU rDNA).Vorticella paraglobosa sp.n.is characterized mainly by its obconical or elongate bell-shaped zooid,C-shaped macronucleus,single ventrally located contractile vacuole,two-rowed infundibular polykinety 3,and 28-38 silverlines between peristome and aboral tro-chal band and 10-15 between aboral trochal band and scopula.Vorticella cotyliformis sp.n.differs from its congeners mainly by its double-layered peristomial lip,cup-shaped zooid,J-shaped macronucleus,single ventrally positioned contractile vacuole,three-rowed infundibular polykinety 3,and 70-85 silverlines between peristome and aboral trochal band and 21-25 between aboral trochal band and scopula.The SSU rDNA sequences of the two new species were obtained,and the subsequent molecular phylogenetic analysis supported their taxonomic classification.

Boehmeria nivea var. strigosa (Urticaceae), a new variety from Southwest China

Boehmeria nivea var.strigosa Zeng Y.Wu&Y.Zhao,a new variety of B.nivea(Urticaceae)from Southwest China,is here described based on evidence from morphology and molecular phylogeny.This new variety is mainly characterized by its green abaxial leaf blade,partly connate stipules,and densely patent strigose hairs on stems and potioles.The phylogenetic analysis based on rbc L,nrDNA and rbc L+nrDNA datasets,revealed that all individuals of B.nivea var.strigosa formed a monophyletic group.The conservation status of B.nivea var.strigosa is assessed as“Near Threatened”(NT)according to IUCN evaluation criteria.The discovery of this new variety is not only crucial for the taxonomy of ramie,but also provides reference for the exploration and utilization of ramie.

A New Species of the Genus Boulenophrys(Anura,Megophryidae)from Eastern Guangdong,China

A new horned toad species,Boulenophrys elongata sp.nov.,is described from Mt.Lianhua,Huidong County,eastern Guangdong,China.The new species is phylogenetically closest to B.brachykolos,which is restricted to the east of the Pearl River Estuary(i.e.,Shenzhen City and Hong Kong SAR).The new species can be distinguished from all recognized congeners by a combination of morphological characters and genetic divergences in the combined mitochondrial 16S,12S and CO1 genes.We recommend the new species be listed as Data Deficient(DD)in the IUCN categorization,pending further investigation.

A new genus of Asiatic short-tailed shrew (Soricidae, Eulipotyphla) based on molecular and morphological comparisons

Blarinellini is a tribe of soricine shrews comprised of nine fossil genera and one extant genus. Blarinelline shrews were once widely distributed throughout Eurasia and North America, though only members of the Asiatic short-tailed shrew genus Blarinella currently persist （mostly in southwestern China and adjacent areas）. Only three forms of Blarinella have been recognized as either species or subspecies However, recent molecular studies indicated a strikingly deep divergence within the genus, implying the existence of a distinct genus-level lineage. We sequenced the complete mitochondrial genomes and one nuclear gene of three Asiatic short-tailed and two North American shrews and analyzed them morphometrically and morphologically. Our molecular analyses revealed that specimens ascribed to B. griselda formed two deeply diverged lineages, one a close relative to B. quadraticauda, whereas the other--comprised of topotype specimens from southern Gansu-iverged from other Blarinella in the middle Miocene （ca. 18.2 million years ago （Ma）, 95% confidence interval=13.4-23.6 Ma）. Although the skulls were similarly shaped in both lineages, we observed several diagnostic characteristics, including the shape of the upper p4. In consideration of the molecular and morphological evidence, we recognize B. griselda as the sole species of a new genus, namely, Pantherina gen. nov. Interestingly, some characteristics of Pantherina griselda are more similar to fossil genera, suggesting it represents an evolutionarily more primitive form than Blarinella.Recognition of this new genus sheds light on the systematics and evolutionary history of the tribe Blarinellini throughout Eurasia and North America.

Culture observation and molecular phylogenetic analysis on the blooming green alga Chaetomorpha valida (Cladophorales, Chlorophyta) from China

The marine green alga Chaetomorpha valida fouls aquaculture ponds along the coastal cities of Dalian and Rongcheng, China. Unialgal cultures were observed under a microscope to determine the developmental morphological characters of C. valida. Results reveal that gametophytic filaments often produce lateral branches under laboratory culture conditions, suggesting an atypical heteromorphic life cycle of C. valida between unbranched sporophytes and branched gametophytes, which differs from typical isomorphic alternation of Chaetomorpha species. The shape of the basal attachment cell, an important taxonomic character within the genus, was found variable depending on environmental conditions. The 18S rDNA and 28S rDNA regions were used to explore the phylogenetic affinity of the taxa. Inferred trees from 18S rDNA sequences revealed a close relationship between C. valida and Chaetomorpha moniligera. These results would enrich information in general biology and morphological plasticity of C. valida and provided a basis for future identification of green tide forming algae.

Phylogeny of Ptychostomum (Bryaceae,Musci) inferred from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast rps4

The phylogeny of Ptychostomum was first spacer （ITS） region of the nuclear ribosomal （nr） DNA DNA rps4 sequences. Maximum parsimony, maximum undertaken based on analysis of the internal transcribed and by combining data from nrDNA ITS and chloroplast likelihood, and Bayesian analyses all support the conclusion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii （Schwagr.） J. R. Spence （≡ Bryum funkii Schwaigr.） is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well-supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with transferring B. amblyodon Mull. Hal. （≡ B. inclinatum （Brid.） Turton≡ Bryum archangelicum Bruch ＆ Schimp.）, Bryum lonchocaulon Mull. Hal., Bryum pallescens Schleich. ex Schwaigr., and Bryum pallens Sw. to Ptychostomum.

Phylogeny of Limenitidinae Butterflies (Lepidoptera: Nymphalidae) Inferred from Mitochondrial Cytochrome Oxidase I Gene Sequences

The phylogenetic analyses of the subfamily Limenitidinae are performed based on 1 471 bp of mtDNA cytochrome oxidase subunit I （COI） gene sequence data which were obtained from 21 individuals spanning 9 genera, along with those of 17 species obtained from GenBank, using Apatura iris, Aglais urticae, and Polyura dolon as outgroup species. Although the transitions at the third codon positions of the COI data set were highly saturated, they were still retained for analysis as they contain the majority of the phylogenetic information, and thus, the maximum pasimony （MP） under different weighting schemes and maximum likelihood （ML） trees were reconstructed in this study. The results showed that within this subfamily, the results based on the COI gene sequences are approximately identical to the traditional classification results. However, the clustering of Lexias pardalis and Tanaecia julii within the genus Euthalia as well as the clustering of Phaedyma aspasia within the genus Neptis with weak support are different from that of the current classification scheme made by Chinese scholars. The genus Limenitis is splited into two subclusters in the trees constructed by using MP and ML methods. These results support one of the strongest hypotheses for the tribe relationships within Limenitidinae.