The Problem of Characters Susceptible to Parallel Evolution in Phylogenetic Reconstructions: Suggestion of a Practical Method and Its Application to Cave Animals

We here propose a procedure to treat characters which are susceptible to parallel evolution (in this case, troglomorphisms) as a replacement for the two procedures used so far: to either completely consider or completely disregard these characters. These procedures may lead to one of two opposite errors, respectively, (1) to consider them as true synapomorphies when they are not or (2) to disregard them as true synapomorphies when they are. We suggest herein to recode the characters by splitting each troglomorphic character into as many as the number of taxa which show the given troglomorphic state. For each split character each taxon will have the derived state, while the others will be coded as missing data. We provide three real examples to test our procedure and conclude that it may give results different from those of the other two procedures. This means that our procedure does not repeat the two above "errors." In addition, we believe that the procedure filters the possible biases, resulting in trees in which the troglomorphisms do have phylogenetic signals.     

Synthetic two-piece and three-piece split inteins for protein trans-splicing

Inteins are protein-intervening sequences that can self-excise and concomitantly splice together the flanking polypeptides. Two-piece split inteins capable of protein trans-splicing have been found in nature and engineered in laboratories, but they all have a similar split site corresponding to the endonuclease domain of the intein. Can inteins be split at other sites and do trans-splicing? After testing 13 split sites engineered into a Ssp DnaB mini-intein, we report the finding of three new split sites that each produced a two-piece split intein capable of protein trans-splicing. These three functional split sites are located in different loop regions between beta-strands of the intein structure, and one of them is just 11 amino acids from the beginning of the intein. Because different inteins have similar structures and similar beta-strands, these new split sites may be generalized to other inteins. We have also demonstrated for the first time that a three-piece split intein could function in protein trans-splicing. These findings have implications for intein structure-function, evolution, and uses in biotechnology.     

Taxonomic continuum of Alchemilla (Rosaceae) in Estonia

23 widespread apomictic Alchemilla microspecies occurring in Estonia are analyzed to investigate whether the species and higher rank taxa are distinct, how variable these taxa are and which characters distinguish them better. Cluster analysis, discriminant analysis, analysis of variance, principal component analysis and continuum analysis are used for data processing. The characters form four correlative groups, describing (i) vegetative and (ii) generative parts of the plant body, (iii) hairiness characters and, (iv) leaf teeth measurements. The best characters according to analysis of variance for disünguishing species are hairiness characters, but often they distinguish only few species very clearly and cannot be used for the remaining ones. Hence the other characters cannot be excluded. From the studied species only A. plicata, A. semilunaris and A. lindbergiana are significantly distinct from all others. The remaining ones form a complicated network of mutually indistinct pairs. Higher rank taxa — sections and series according to Rothmaler, Fröhner and Yuzepchuk are better separated, containing very few mutually indistinct pairs. Results from species centroids' clustering are most congruent with Fröhner's system, but still some changes seem to be necessary and a corrected system is proposed here. Section Plicatae is split into two series: Pubescentes and Barbulatae , sections Alchemilla, Ultravulgares and Decumbentes are joined as three series of section Hirsutae , and A. filicaulis is moved from section Plicatae to section Coriaceae. Coriaceae should also be split into three series: Exuentes (A. filicaulis), Glabricaules (A. glabricaulis) and Coriaceae (other species).     

Diversity of developmental patterns in achelate lobsters—today and in the Mesozoic

Modern achelate lobsters, slipper and spiny lobsters, have a specific post-embryonic developmental pattern with the following phases: phyllosoma, nisto (slipper lobsters) or puerulus (spiny lobsters), juvenile and adult. The phyllosoma is a peculiar larva, which transforms through a metamorphic moult into another larval form, the nisto or puerulus which largely resembles the juvenile. Unlike the nisto and puerulus, the phyllosoma is characterised by numerous morphological differences to the adult, e.g. a thin head shield, elongate appendages, exopods on these appendages and a special claw. Our reinvestigation of the 85 million years old fossil “Eryoneicus sahelalmae” demonstrates that it represents an unusual type of achelatan lobster larva, characterised by a mixture of phyllosoma and post-phyllosoma characters. We ascribe it to its own genus: Polzicaris nov. gen. We study its significance by comparisons with other cases of Mesozoic fossil larvae also characterised by a mixture of characters. Accordingly, all these larvae are interpreted as ontogenetic intermediates between phyllosoma and post-phyllosoma morphology. Remarkably, most of the larvae show a unique mixture of retained larval and already developed post-larval features. Considering the different—and incompatible—mixture of characters of each of these larvae and their wide geographical and temporal distribution, we interpret all these larvae as belonging to distinct species. The particular character combinations in the different larvae make it currently difficult to reconstruct an evolutionary scenario with a stepwise character acquisition. Yet, it can be concluded that a larger diversity of larval forms and developmental patterns occurred in Mesozoic than in modern faunas.     

Morphological and molecular diversity of the monocercomonadid genera Monocercomonas, Hexamastix, and Honigbergiella gen. nov

The family Monocercomonadidae (Parabasala, Trichomonadida) is characterized by the absence of a costa and in most species also of an undulating membrane; both of which are typical structures of trichomonadids. We have examined 25 isolates of Monocercomonadidae species by sequencing of the SSU rDNA and the ITS region and by light and transmission electron microscopy. The isolates formed three distinct phylogenetically unrelated clades: (1) Monocercomonas colubrorum, (2) Monocercomonas ruminantium together with a strain ATCC 50321 designated as Pseudotrichomonas keilini, and (3) Hexamastix. Twenty isolates of Monocercomonas colubrorum split into three clades with no host-specificity. The morphological differences among clades were insufficient to classify them as a separate species. Non-monophyly of the cattle commensal Monocercomonas ruminantium with the type species Monocercomonas colubrorum and absence of Pseudotrichomonas characters in the free-living strain ATCC 50321 led to their reclassification into a new genus (Honigbergiella gen. nov.). The close relationship of these strains indicates a recent switch between a free-living habit and endobiosis. Two strains of Hexamastix represented different species -Hexamastix kirbyi Honigberg 1955 and Hexamastix mitis sp. nov. Polyphyly of the Monocercomonadidae confirmed that the absence of a costa and an undulating membrane are not taxonomically significant characters and were probably secondarily lost in some or all clades. Our observations, however, indicated that other characters - infrakinetosomal body, comb-like structure, marginal lamella, and the type of axostyle - are fully consistent with the position of Monocercomonadidae species in the parabasalian tree and are, therefore, reasonable taxonomic characters.     

The origin of photic behavior and the evolution of sexual communication in fireflies (Coleoptera: Lampyridae)

Through a phylogenetic analysis using adult morphological characters, we show that the origin of bioluminescence in cantharoid beetles appears to predate the origin of the family Lampyridae. The ability to produce and emit photic signals was first gained by larvae and appears to function as an aposematic warning display; it was subsequently gained in adults and is used as a sexual signal. Our analysis also suggests that while pheromonal sexual signals are used basally in the family, they are used in conjunction with and then subsequently replaced by photic signals in some lampyrid lineages. Both photic signals and the photic organs used to produce them have become greatly elaborated in the fireflies that no longer employ pheromonal sexual signals. In addition, the ability to produce a flashed sexual signal appears to have arisen at least three times in the family Lampyridae. Convergent evolution is also evident in a number of adult male photic organ morphologies. Further, we recommend that individual signal system components be compared rather than overall signal system complexity. The use of this strategy may allow one to recognize and better interpret adaptive correlations despite convergence or loss. We demonstrate that phylogenetic analysis is a powerful tool even for rapidly evolving traits.     

Evidence for kinship between diverse G-protein coupled receptors.

In an earlier publication we described similarities at the primary sequence level between the first probable plant G-protein coupled receptor (GPCR) and three GPCR families (families A, B and F according to Kolakowski's classification) that were previously considered evolutionarily unrelated. Here we analyze further the relatedness among different GPCR families.By using PSI-BLAST, which is a search algorithm that is more potent in detecting weak similarities, one finds additional similarities between GPCR families that have not previously been described. Based on these comparisons, it is possible to divide all the GPCR families into one large clade and two smaller ones. The large clade includes the rhodopsin family (family A), the glucagon receptor family (family B), cyclic AMP receptors (family F), an Arabidopsis thaliana receptor, the Frizzled family and probably also the STE3 pheromone receptors (family E) and vomeronasal receptors type 1. The smaller clades consist of, in one case, BOSS and the GABA-B family of receptors (family C), and in the other the STE2 pheromone receptors (family D) alone. Although our findings are likely to reflect a common ancestry within each of these clades, whether or not two or all three of the clades also share an even more ancient ancestor between them remains an open question that cannot be answered from our present data.     

Identification of related proteins on family, superfamily and fold level

Proteins might have considerable structural similarities even when no evolutionary relationship of their sequences can be detected. This property is often referred to as the proteins sharing only a "fold". Of course, there are also sequences of common origin in each fold, called a "superfamily", and in them groups of sequences with clear similarities, designated "family". Developing algorithms to reliably identify proteins related at any level is one of the most important challenges in the fast growing field of bioinformatics today. However, it is not at all certain that a method proficient at finding sequence similarities performs well at the other levels, or vice versa.Here, we have compared the performance of various search methods on these different levels of similarity. As expected, we show that it becomes much harder to detect proteins as their sequences diverge. For family related sequences the best method gets 75% of the top hits correct. When the sequences differ but the proteins belong to the same superfamily this drops to 29%, and in the case of proteins with only fold similarity it is as low as 15%. We have made a more complete analysis of the performance of different algorithms than earlier studies, also including threading methods in the comparison. Using this method a more detailed picture emerges, showing multiple sequence information to improve detection on the two closer levels of relationship. We have also compared the different methods of including this information in prediction algorithms.For lower specificities, the best scheme to use is a linking method connecting proteins through an intermediate hit. For higher specificities, better performance is obtained by PSI-BLAST and some procedures using hidden Markov models. We also show that a threading method, THREADER, performs significantly better than any other method at fold recognition.     

Identification of family relationships by epigenetic traits

In analyzing family burials, it is often necessary to establish the nature of the family relationship. This study examines 18 skulls from the 14th and 15th century, presumptively assigned to the family of the Counts of Celje from the territory of present-day Slovenia. Though DNA analysis is the identification method of choice, it is not always possible to apply it. In our case we were unable to isolate the genetic material and had to use classical identification methods. After determining sex and age, we established the kinship by comparison of X-ray images of paranasal cavities (frontal and maxillary sinuses, and also orbital and nasal cavities), the shape and size of which are autosomal dominant inherited characters. The comparison also extends to numerous other likewise inherited epigenetic trait similarities on the skulls. We identified all skulls, compared them with historical and genealogical data, grouped them into nuclear families--one of them is presented in this article-, and proved that they belong to the assigned family. This multidisciplinary work, based on latest epigenetic research, is highly relevant for modern non-genetic identification studies and verification of kinship by skulls collectively interred and can be applied in similar cases where isolation of DNA is no longer possible, even though the skeletal remains may not be old.     

Conservative evolution in duplicated genes of the primate Class I ADH cluster

Humans have seven alcohol dehydrogenase genes (ADH) falling into five classes. Three out of the seven genes (ADH1A, ADH1B and ADH1C) belonging to Class I are expressed primarily in liver and code the main enzymes catalyzing ethanol oxidization. The three genes are tandemly arrayed within the ADH cluster on chromosome 4 and have very high nucleotide similarity to each other (exons: >90%; introns: >70%), suggesting the genes have been generated by duplication event(s). One explanation for maintaining similarity of such clustered genes is homogenization via gene conversion(s). Alternatively, recency of the duplications or some other functional constraints might explain the high similarities among the genes. To test for gene conversion, we sequenced introns 2, 3, and 8 of all three Class I genes (total>15.0 kb) for five non-human primates--four great apes and one Old World Monkey (OWM)--and compared them with those of humans. The phylogenetic analysis shows each intron sequence clusters strongly within each gene, giving no evidence for gene conversion(s). Several lines of evidence indicate that the first split was between ADH1C and the gene that gave rise to ADH1A and ADH1B. We also analyzed cDNA sequences of the three genes that have been previously reported in mouse and Catarrhines (OWMs, chimpanzee, and humans) and found that the synonymous and non-synonymous substitution (dN/dS) ratios in all pairs are less than 1 representing purifying selection. This suggests that purifying selection is more important than gene conversion(s) in maintaining the overall sequence similarity among the Class I genes. We speculate that the highly conserved sequences on the three duplicated genes in primates have been achieved essentially by maintaining stability of the hetero-dimer formation that might have been related to dietary adaptation in primate evolution.     

Fifty years of character compatibility concepts at work

In the mid 19th century,systematic biologists realized that observable similarities and differences among a group of related species could be the basis for hypotheses about the evolutionary relationships among the species and their ancestors.Such hypotheses Can be expressed as characters.A character is comprised of two or more character states of species considered to be similar with respect to a basis for comparison.The states of a character may also be arranged into a character state tree to hypothesize speciation events associated with changes from one character state to another.In the mid 20th century.some systematists realized that sometimes paxrs of characters(or character state trees)could be incompatible as hypotheses,i.e.,they could not both be true.Through the 1950s,'60s and'70s,tests for,and ways to resolve,incompatibilities were used to estimate an ancestor relation based on mutually compatible characters.An estimate was often shown as a diagram connecting ancestors to their immediate descendants(not quite correctly)called a phylogenetic tree.More recently,other applications of compatibility concepts have been developed,including:identify characters that appear to be random in the context of their data set;combine estimates of ancestor relations for subsets of taxa in a larger collection into a single estimate(a so-called supertree)for the whole collection;and interpret geographic patterns in an evolutionary context.     

浙江天童常绿阔叶林藤本植物的适应生态学I. 叶片解剖特征的比较

利用解剖学方法对浙江天童森林公园常绿阔叶林的 10属 11种木质藤本叶片的解剖特征进行了比较和因子分析 ,结果表明 :藤本植物叶片结构在不同种类之间具有明显的差异 ,同种藤本的不同植物体的叶片也存在一定的差异 ;这些差异除受遗传因子控制外 ,环境因子 (主要为光照和水分 )也有重要作用 ;攀援方式对藤本叶片特征有一定影响 ,但未表现出规律性。尽管叶片的基本结构仍属中生类型 ,但也表现出一定的趋异适应 ,根据叶片适应特征的差异 ,11种藤本植物可分为 3种类型 ,即偏旱生或阳生叶类型 (包括薜荔 (Ficus pumila)、珍珠莲 (Ficussarmentosa var. henry)和藤构 (Broussonetia kaempferi) ) ,偏阴生叶类型 (羊角藤 (Morinda umbellata)和昆明鸡血藤(Millettia reticulata) )和中生叶类型 (广东蛇葡萄 (Ampelopsis cantoniensis)、中华常春藤 (H edera nepalensis var.sinensis)、短梗南蛇藤 (Celastrus rosthonianus)、暗色菝葜 (Smilax lanceiofolia var.opaca)、薯莨 (Dioscorea cirrhosa)和香港黄檀 (Dalbergia millettii) )。藤本叶片的各部分结构特征对生境条件的适应并未表现出一致的同步变化 ,存在着一定的差异或可塑性 ,这可能由于藤本生长环境经常发生变动所至     

Alternative phenotypes of male mating behaviour in the two-spotted spider mite

Severe intraspecific competition for mates selects for aggressive individuals but may also lead to the evolution of alternative phenotypes that do not act aggressively, yet manage to acquire matings. The two-spotted spider mite, Tetranychus urticae, shows male mate-guarding behaviour and male–male combat for available females. This may provide opportunity for weaker males to avoid fighting by adopting alternative mating behaviour such as sneaker or satellite tactics as observed in other animals. We investigated male precopulatory behaviour in the two-spotted spider mite by means of video-techniques and found three types of male mating behaviour: territorial, sneaker and opportunistic. Territorial and sneaker males associate with female teleiochrysales and spend much time guarding them. Territorial males are easily disturbed by rival males and engage themselves in fights with them. However, sneaker males are not at all disturbed by rival males, never engage in fights and, strikingly, never face attack by territorial males. Opportunistic males wander around in search of females that are in the teleiochrysalis stage but very close to or at emergence. To quickly classify any given mate-guarding male as territorial or sneaker we developed a method based on the instantaneous response of males to disturbance by a live male mounted on top of a brush. We tested this method against the response of the same males to natural disturbance by two or three other males. Because this method proved to be successful, we used it to collect territorial and sneaker males, and subjected them to morphological analysis to assess whether the various behavioural phenotypes are associated with different morphological characters. However, we found no statistical differences between territorial and sneaker males, concerning the length of the first legs, the stylets, the pedipalps or the body.     

Ecological polarity in primate tion

Relationships of the catarrhine primates based on morphological similarity are presented and then combined with habitat data to determine polarity of ecological change in the catarrhines. The ancestral habitat of two sister-groups is estimated by combining that of the sister groups, so that if they share one habitat type the ancestral condition is taken to be that type, or if they differ the ancestral condition is taken to be both alternatives. Analysed in this way the ancestral habitat preference of the Catarrhini is tropical lowland forest, and while the Hominoidea retain this primitive condition, the Cercopithecoidea are derived with a savanna habitat preference. Most hominoids retain the primitive forest habitat condition, and those groups that are associated with woodland-savanna habitats, notably the ramapitbecines and hominines, therefore share a derived habitat preference. There is no evidence, however, that this arose through common ancestry, and it is concluded that the functional similarities between the two groups could have arisen through parallel adaptation to the same habitat type. Similarly, many of the extant cercopithecoid groups that are now forest living may have re-entered forest independently. The derived savanna habitat preference of the cercopithecoids is linked with a number of morphological characters that also are derived with respect to catarrhine ancestry, and these include their terrestrial and/or their above-branch adaptations, and their specialized digestive, dietary and dental adaptations. In contrast, the hominoids that retain the primitive catarrhine habitat preference also retain the primitive condition in all these characters.     

Comparative myology of the forelimb of Liolaemus sand lizards (Liolaemidae)

The lizard genus Liolaemus includes numerous constituent clusters of putatively related taxa, one of which is the Liolaemus boulengeri group, which in turn includes the sand lizards (of the Liolaemus wiegmannii subgroup). Members of the sand lizard group exhibit three different modes of burying into sand. The general morphology of the forelimb muscles of those Liolaemus species is analysed. Herein, we present a study of the forelimb musculature of all species considered by Halloy et al. (1998). This study has three principal goals. First, we are seeking myological characters that will be useful in formulating phylogenetic hypothesis about the species of Liolaemus. With these characters, we also wish to compile morphological data that represent the morphological space implied in the diverse locomotor behaviours of these animals. Second, we are looking for derived features that reflect functional changes in the use of forelimb. Third, we wish to provide a cladistic analysis that can be used to test phylogenetic hypothesis derived from other sources of data. We present 48 characters in a data set and analyse it cladistically. We obtained a hypothesis of relationships of the Liolaemus species and compared this with previous hypotheses based on other characters. The trees obtained are not congruent with previously proposed phylogenies. We were unable to identify in our trees nodes that are based on structures reflecting functional changes in the use of the forelimb. The morphological similarities in the forelimb musculature of all species analysed seems to conform a very conservative general anatomical pattern with which Liolaemus sand lizards perform most of their locomotor behaviours.     

Comparative anatomy of conifer leaves

The epidermis and internal structure of 103 species and 8 varieties of conifer leaves belonging to 37 genera of 7 families have been comparatively investigated. The anatomical characters of leaves of all the genera (or sections) studied are described systematically and a key to the genera (or sections) is also presented. It is shown that in order to identify at the genus level, the leaf types of LaubenFels’ may be used as the primary characterization. And it can also be seen that the following characters which are relatively constant at the genus level provide an important basis for identification: the epidermal structure, the presence or absence of hypodermis, the degree of differentiation of mesophyll, the occurrence of vascular bundle sheath or endodermis, the transfusion tissue type, the number and relative spatial position of vascular bundles, the number and distribution of resin canal and the presence of sclereid or sclerenchyma. The genera of Cupressaceae (except for Juniperus and Sabina) are of scale leaves (Leaf type-III) with little differences in structural characters. It’s difficult to distinguish them from each other. In addition, some problems concerning the significance of the resin canal, the endodermis with Casparian-strip, the sclerenchyma and the vascular bundle in identificationare here also discussed.     

浙江天童常绿阔叶林藤本植物的适应生态学：Ⅰ、叶片解剖特征的比较

利用解剖学方法对浙江天童森林公园常绿阔叶林的１０属１１种木质藤本叶片的解剖特征进行了比较和因子分析,结果表明：藤本植物叶片结构在不同种类子之间具有明显的差异,同种藤本的不同植物的叶片也存在一定的差异;这些差异除受遗传因子控制外,环境因子（主要为光照和水分）也有重要作用;攀援方式对藤本叶片特征有一定影响,但未表现出规律性。尽管叶片的基本结构仍属中生类型,但也表现出一定的趋异适应,根据叶片适应特征的差异,１１种藤本植物可分为３种类型,即偏旱生或阳生叶类型（包括薜荔（Ｆicus pumila)、珍珠莲（Ficus sarmentosa var.henry)和藤构（Ｂroussonetia kaempferi)),偏阴生叶类型（羊角藤（Morinda umbellata)和昆明鸡血藤（Millettia reticulata）中和生叶类型（广东蛇葡萄（Ａmpelopsis cantoniensis)、中华常春藤（Hedera nepalensis var.sinensis)、短梗南蛇藤（Celastrus rosthoniauns)、暗色菝葜（Smilax lanceiofolia var.opaca）、薯莨（Ｄioscorea cirrhosa)和香港黄檀（Ｄalbergia millettii)。藤本叶片的各部分结构特征对生境条件的适应并未表现出一致的同步变化,存在着一定的差异或可塑性,这可能由藤本生长环境经常发生变动所致。     

Evidence of a reproductive barrier between two forms of the marine periwinkle Littorina fabalis (Gastropoda)

Studies of allozyme variation may reveal unexpected patterns of genetic variation which challenge earlier conclusions of species delimitations based on morphological data. However, allozyme variation alone may not be sufficient to resolve this kind of problem. For example, populations of the marine intertidal snail Littorina fabalis (=Littorina mariae) from wave exposed parts end from protected parts of the same shores are distinguished by different alleles of arginine kinase (Ark) while indifferent, or very nearly so, in another 29 loci. Intermediate populations have large deficiencies of exposed/sheltered heterozygote classes of Ark and we have earlier suggested habitat-related selection in this locus as the explanation. In this study we estimated growth rate of individual snails of different Ark-genotypes in three different habitats (exposed, sheltered and intermediate). In all habitats the snails homozygous for alleles of ‘exposed’ type grew faster and matured at a larger size than did snails homozygous for alleles of ‘sheltered’ types. This relationship was indirectly confirmed in three additional sites of intermediate exposure where exposed AA-genotypes dominated among large (>8 mm) snails while the sheltered genotypes dominated among small (<5 mm) snails of truly sympatric samples. We furthermore found small differences in allele frequencies of two other loci (Pgi and Pgm-2) and in shell colour frequencies, comparing sympatric snails of exposed and sheltered Ark-homozygotes. Although we found no signs of habitat-related selection among snails of different Ark-genotype, or selection against heterozygotes, we cannot reject selection in Ark, as our experiments only covered one island, one season and grown-up snails. The coupling between allozyme and phenotypic characters in strictly sympatric samples of snails suggests the presence of two gene pools. Perhaps the large and small forms of L. fabalis represent very closely related cryptic taxa. However, introgression between them seems a possible explanation for the striking similarities in the vast majority of morphological and allozyme characters.     

Phylogenomic analysis resolves the relationships among net‐winged beetles (Coleoptera: Lycidae) and reveals the parallel evolution of morphological traits

Net‐winged beetles (Coleoptera: Lycidae) are a diverse group of elateroids known for aposematism and neoteny. Phylogenetic analyses of morphological and molecular data have revealed different results with respect to within‐group relationships. In this study, we recovered a highly supported phylogenomic phylogeny and identified seven subfamilies: Dexorinae stat.n. , Calochrominae stat.n. , Erotinae, Ateliinae, Lycinae, Lyropaeinae stat.n. and Metriorrhynchinae stat.n. Our results suggest that female neoteny evolved multiple times. Therefore, the development of similar morphological modifications in neotenics may be linked and may have produced characteristics such as body miniaturization, structural simplification, i.e. reduction of mouthparts, fewer antennomeres and palpomeres, uniquely shaped terminal palpomeres, shortened elytra, the loss of coadaptation between the elytra and pronotum, and others. Additional traits evolved in parallel due to similarities in biology, function and sexual selection. These characteristics include mimetic similarities, the presence of the rostrum, pronotal carinae and elytral costae, and the structure of male genitalia. By comparing the phylogenomic topology with the evolution of morphological characters, we were able to identify evolutionary trends in lycids and compare them with analogues for other neotenic elateroids. These traits have not been accepted as homoplasies due to the ambiguous phylogenetic signal from Sanger sequencing markers.