Partial Cytochrome Oxidase Research Articles

Old Aegean palaeoevents driving the diversification of an endemic isopod species (Oniscidea, Trachelipodidae)

The Greek endemic isopod species Trachelipus aegaeus is distributed in Aegean islands and the adjacent coastal parts of the Greek mainland. Major palaeogeographic events of the Aegean archipelago, such as the formation of the mid‐Aegean trench and the Messinian Salinity Crisis, have been often employed as major causal factors of evolutionary events and phylogeographic patterns exhibited by several taxa. Herein, we infer phylogenetic relationships among T. aegaeus populations using partial cytochrome oxidase subunit I (COI) and 16S rRNA sequences. Due to the poor preservation of the specimens, we propose a modified DNA extraction protocol, which returned highly positive results in terms of the quality of the total extracted DNA. We implement a calibrated molecular clock and path sampling analysis, using alternative palaeogeographic events and rates of substitution, to evaluate the biogeographic history of the species and to estimate the chronology of diversification events among its populations. Our results are clearly in favour of the scenario of the MAT triggering vicariance among most T. aegaus populations. Moreover, the large intraspecific genetic divergence (0–19% for COI and 0–20.3% for the 16S rRNA) and the overall phylogeographic patterns depicted herein seem not to have been obscured by more recent palaeogeological events. A role of dispersal, probably human‐aided, is assumed for certain ‘deviant’ cases.

Vinodolia fiumana Radoman, 1973 (Caenogastropoda: Rissooidea): rediscovery and relationships of a species presumed extinct

Vinodolia fiumana was described by Radoman from Glogi spring in 1973, and later not found, thus considered extinct. In the CLECOM list Anagastina, Dalmatinella and Prespiana were classified, as subgenera, within the genus Vinodolia. After visiting the Glogi spring on several occasions, we found four specimens of V. fiumana. Its shell, soft part external morphology, radula, penis and female reproductive organs are described. Partial cytochrome oxidase subunit I mitochondrial and 18S rRNA nuclear gene sequences were used to infer phylogenetic relationships of Vinodolia. Sadleriana was found to be the sister taxon of Vinodolia, but neither Anagastina (closely related with Radomaniola), nor Dalmatinella were closely related with it.

Moisture variables, and not temperature, are responsible for climate filtering and genetic bottlenecks in the South African endemic terrestrial mollusc Prestonella (Orthalicoidea)

Non-vagile taxa such as terrestrial molluscs are susceptible to stochastic environmental events that can cause local extinctions or population declines, and extinctions of terrestrial mollusc species are among the highest documented. Many terrestrial snails are habitat specialists, and genetic studies using both allozyme and DNA sequence data have indicated that many species contain substantial and geographically structured genetic variation. In this study, we assess the genetic variation within two species of the rare terrestrial snail genus Prestonella, an inhabitant of rocky areas along water courses in the southern Great Escarpment of South Africa, and correlate genetic diversity to climatic variables. DNA sequence data from mitochondrial 16S rDNA and partial cytochrome oxidase I genes indicate that neither species is monophyletic, and that populations are deeply divergent, even over distances of a few hundred metres. Principal components anaylsis of climatic variables derived from two databases indicates that genetic diversity of the populations is correlated to moisture-related climatic variables. Populations with little or no diversity occur in more arid regions, and are thus most at risk from any future climatic changes that would increase aridification. These moisture variables are thus potent drivers of genetic bottlenecks and may have resulted in historical climate filtering or limitation of the distribution of these species. Temperature appears to be a less important variable, a finding supported by physiological data based on heart rates that show that death occurs only at temperatures far higher than found in their environment.

Phylogeographic analysis of Apis cerana populations on Hainan Island and southern mainland China, based on mitochondrial DNA sequences

The phylogeography of the eastern honeybee (Apis cerana) in southern China is poorly understood. In the present study, we investigated phylogenetic relationships among A. cerana populations on Hainan island and southern mainland China. Analyses were based on the partial cytochrome oxidase subunit 1 gene of mitochondrial DNA and A. cerana samples representing 12 geographic populations. We identified 57 novel haplotypes (GenBank accession numbers: KC175488-KC175544) within our sampled populations and inferred two deeply divergent intraspecific lineages when previously published sequences were included, which could be further divided into six sublineages. We also found a significant phylogeographic structure within lineage A. Our results reveal that A. cerana populations on Hainan island and southern mainland China have undergone a long period of differentiation in isolation followed by historical demographic expansion. Although gene flow now occurs between these locations, gene flow has occurred over comparatively a short time period, and is likely to be the result of anthropogenic effects.

Epinephelus moara: a valid species of the family Epinephelidae (Pisces: Perciformes)

This study documents the major external and internal morphological differences between Epinephelus bruneus and Epinephelus moara, and analyses the complete mitogenomes of both species. The partial cytochrome oxidase subunit I (coI) sequence divergence between E. bruneus and E. moara is significantly higher than specimens within the same species (P < 0·05). Analyses of gene flow (Nm = 0·02) and genetic differentiation (ϕst = 0·92995, P > 0·05) reveal reproductive isolation between E. bruneus and E. moara. These results support the hypothesis that E. moara is a valid species. Further molecular comparisons between E. bruneus and E. moara obtained in this study and a specimen identified in GenBank as E. bruneus from South Korea reveal that the latter is identical to E. moara rather than to E. bruneus.

Application of Cytochrome Oxidase I Sequences for Phylogenetic Analysis and Identification of Thrips Species Occurring on Vegetable Crops

Thrips are direct pests as well as vectors of important viruses infecting crop plants. One of the major constraints in studying the relationship between thrips vectors and tospoviruses is the difficulty of identifying the vector species because of high intraspecific variation among thrips populations. Molecular approaches have been used to identify species differences. In this study, partial cytochrome oxidase I (COI) sequences were used to understand the phylogenetic relationship among thrips populations, and assess their usefulness to identify and classify unknown thrips species collected from different crops. In total, 29 COI variants were obtained while examining the sequence polymorphisms in COI of 182 insects analyzed in this study, which were collected from six countries on tomato, chilli, onion, cabbage, cucumber, watermelon, Ethiopian mustard, French bean, and peanut. The phylogenetic analysis showed that the insects used in this study clustered with five distinct species-groups designated as Thrips palmi group, T. tabaci group, Frankliniella occidentalis group, Scirtothrips dorsalis group and an unclassified group. Higher intraspecific genetic variation was observed in S. dorsalis and T. palmi followed by T. tabaci and F. occidentalis. Thus, it was confirmed that the COI gene could be useful in grouping different thrips species and genera that coexist in a particular cropping system.

Genetic distinctiveness of the greater long-tailed hamster, Tscherskia triton nestor (Rodentia: Mammalia), from Jeju Island, Korea: cytochrome oxidase I and cytochrome b sequence analyses

To examine the taxonomic status of Tscherskia triton nestor, which inhabits in Korea, we first obtained partial cytochrome oxidase I (COI) (657 bp) and cytochrome b (426 bp) sequences of T. t. nestor from the Korean Jeju Island and Tscherskia triton fuscipes from northeastern China, and these sequences were compared with the corresponding sequences of Tscherskia triton, available from GenBank. In the COI analysis, T. t. nestor from Jeju was distinct from T. t. fuscipe from northeastern China, with the average Jukes–Cantor distance of 3.45%, and in the cytochrome b analysis, T. t. nestor from Jeju was divergent from T. t. fuscipes from northeastern China and T. t. triton from the North China Plain, with the average distance of 3.88%. We conclude that these sequencing results on the distinctiveness of T. t. nestor from Jeju support the current classification of T. t. nestor from Korea, although further analyses with mitochondrial sequences of T. t. nestor from the mainland Korea and nuclear sequences of T. triton from Korea and China are necessary to confirm our findings. In addition, we considered genetically distinct T. t. triton from Jeju as an allopatric phylogroup, which needs special protection for its conservation. Additionally, we found the lack of cytochrome b sequences between T. t. fuscipes from northeastern China and T. t. triton in the North China Plain, although further systematic analyses to clarify the taxonomic status of T. t. fuscipes are necessary.

Taxonomic rank of Indian tortoise: revisit with DNA barcoding perspective

Taxonomic rank of terrestrial tortoise inhabiting in India and adjacent countries has been an unresolved issue. Wild living species of Manouria and Indotestudo genera have been classiied through conventional taxonomy, but not adequately discriminated by mitochondrial cytochrome b gene. Cytochrome oxidase (COI) marker has been quite successful to achieve the exact species level information. There needs of an accurate sequence based effort for the extant northeast (NE) Indian tortoises to identify them accurately and to provide locality information. To estimate the Kimura-2-Parameter (K2P) divergences and to construct the NeighbourJoining (NJ) phylogeny, we generated six partial COI sequences derived from expert-identiied tortoise specimens. Both BLASTn and Bold-IDs revealed the deinitive identity of Manouria, whereas the congeners of Indotestudo remain inconclusive by only a two-fold interspeciic divergence gap with the other named Indian tortoise species. The NJ phylogeny readily differentiated the three Indian tortoise genera with their respective conspecies but depicted a nonsynonomous group of Indotestudo congeners. This study shows that the molecular identiication of Manouria emys phayrei, Indotestudo elongata and an allopatric population of Indotestudo forstenii in NE India corresponds correctly with existing morphological key support.

Evidences of increasing risk of dirofilarioses in southern Italy

Given the spread of Aedes albopictus from northern to southern Italy, and the lack of updated data on Dirofilaria infections, this study was carried out to assess the infection risk for dogs and cats in Apulia region. During a 2-year study, 175 A. albopictus female specimens and samples of blood from 427 dogs (309 privately owned dogs and 118 shelter dogs) and 12 cats were collected. All blood samples were subjected to a modified Knott method, to a test for the detection of circulating Dirofilaria immitis antigen, and to a Dirofilaria species-specific real-time PCR for the simultaneous detection of D. immitis and Dirofilaria repens, targeting on partial cytochrome oxidase subunit 1 and internal transcribed spacer-2, respectively. Two abdomen and one thorax pools from A. albopictus were positive for D. immitis, with minimum infection rates of 1.14 and 0.51, respectively, and a probability of a single positive specimen to be infected of P = 0.6% (95% confidence interval (CI) = 0.12-1.73). Out of 439 examined subjects, 22 (5.0%) tested positive for Dirofilaria spp. in at least one diagnostic test. A specific D. immitis infestation rate of 3.5% was found among the privately owned dogs, while shelter dogs tested positive only for D. repens with a prevalence of 3.4%; one cat tested molecularly positive for D. immitis. There was a significantly higher rate of positivity among guard dogs for D. immitis (odds ratio, 6.24, 95% CI, 1.26-25.28; P < 0.05). The increasing risk of D. immitis infection in southern Italy is supported by the noteworthy positivity of A. albopictus populations and the cat. Our data highlight the usefulness to include filarioid infestation in routine diagnosis.

Endemic angiostrongyliasis in the Brazilian Amazon: Natural parasitism of Angiostrongylus cantonensis in Rattus rattus and R. norvegicus, and sympatric giant African land snails, Achatina fulica

Angiostrongylus cantonensis, the rat lungworm, is one etiological agent of eosinophilic meningoencephalitis in humans. This zoonosis is frequently found in Asia and, more recently, in North America, Caribbean Island and northeastern of South America. Until now, research of A. cantonensis in southern, southeastern and northeastern regions of Brazil has been found natural infections only terrestrial and freshwater intermediate snail hosts (Achatina fulica, Sarasinula marginata, Subulina octona, Bradybaena similaris and Pomacea lineate). In this study, we examined the occurrence of helminthes in the synantropic rodents Rattus rattus and Rattus norvegicus in northern Brazil, focusing on the role of these species as vertebrate hosts of A. cantonensis and A. fulica as intermediate host have found natural. Thirty specimens of R. rattus and twelve of R. norvegicus were collected in the Guamá and Jurunas neighborhoods of the city of Belém, in the Brazilian state of Pará, of which almost 10% harbored adult worms in their pulmonary arteries. Sympatric A. fulica were found to be infected by L3 larvae, which experimental infection confirmed to be A. cantonensis. Natural infection of snails and rodents with A. cantonensis was confirmed through morphological and morphometrical analyses of adults and larvae using light microscopy, scanning electron microscopy and molecular sequences of partial Cytochrome Oxidase subunit I. Phylogenetic analyses showed that A. cantonensis isolated from Pará, Brazil is similar to Japan isolate; once these specimens produced a single haplotype with high bootstrap support with Rio de Janeiro isolate. This study confirms that A. cantonensis is now endemic in northern Brazil, and that R. rattus and R. norvegicus act as natural definitive hosts, and A. fulica as the intermediate host of the parasite in this region.

Species boundaries and host range of tortoise mites (Uropodoidea) phoretic on bark beetles (Scolytinae), using morphometric and molecular markers.

Understanding the ecology and evolutionary history of symbionts and their hosts requires accurate taxonomic knowledge, including clear species boundaries and phylogenies. Tortoise mites (Mesostigmata: Uropodoidea) are among the most diverse arthropod associates of bark beetles (Curculionidae: Scolytinae), but their taxonomy and host associations are largely unstudied. We tested the hypotheses that (1) morphologically defined species are supported by molecular data, and that (2) bark beetle uropodoids with a broad host range comprise cryptic species. To do so, we assessed the species boundaries of uropodoid mites collected from 51 host species, across 11 countries and 103 sites, using morphometric data as well as partial cytochrome oxidase I (COI) and nuclear large subunit ribosomal DNA (28S). Overall, morphologically defined species were confirmed by molecular datasets, with a few exceptions. Twenty-nine of the 36 uropodoid species (Trichouropoda, Nenteria and Uroobovella) collected in this study had narrow host ranges, while seven species had putative broad host ranges. In all but one species, U. orri, our data supported the existence of these host generalists, which contrasts with the typical finding that widespread generalists are actually complexes of cryptic specialists.

Genetic Distinctness of Sorex caecutiens hallamontanus (Soricomorpha: Mammalia) from Jeju Island in Korea: Cytochrome Oxidase I and Cytochrome b Sequence Analyses

To examine genetic divergences of two endemic Sorex caecutiens subspecies from Korea (S. c. hallamontanus in Korean Jeju Island and S. c. annexus in the mainland Korean Peninsula), we obtained partial cytochrome oxidase I (COI) sequences (429 bp) and complete cytochrome b sequences (1,140 bp) from the two Korean subspecies, and we compared these sequences to the corresponding sequences of S. caecutiens, obtained from GenBank. We found that Jeju S. c. hallamontanus is one of three clades within S. caecutiens, with an average Jukes-Cantor distance of 1.57% in the COI sequences and the distance of 2.07% and 11 fixed site differences in the cytochrome b sequences, indicating that Jeju S. c. hallamontanus is one endemic subspecies with concordant genetic distinctness, although further analyses with nuclear DNA sequences are necessary to confirm these findings. However, S. c. annexus from the mainland Korean Peninsula was not divergent from S. c. macropygmaeus from northeastern China and adjacent Russia, indicating that S. c. annexus from the mainland Korean Peninsula is another endemic subspecies with only morphological differences, although it is necessary to reexamine the subspecies status of S. c. annexus.

Cryptic species of mites (Uropodoidea: Uroobovella spp.) associated with burying beetles (Silphidae: Nicrophorus): The collapse of a host generalist revealed by molecular and morphological analyses

Uroobovella (Mesostigmata: Uropodoidea: Urodinychidae) species are among the most common mites associated with carrion-feeding Nicrophorus (Silphidae) beetles. Previous taxonomic understanding suggests that a single host generalist, U. nova, disperses and lives with Nicrophorus species worldwide (reported from at least seven host species). Using morphometrics and morphological characteristics, as well as partial cytochrome oxidase I (COI) and the entire internal transcribed spacer 2 (ITS2) markers, we tested whether this apparent generalist is truly a generalist or rather a complex of cryptic species with narrower host ranges. Based on deutonymph mites collected from 14 host species across six countries and 17 provinces or states, we show that U. nova represents at least five morphologically similar species with relatively restricted host ranges. Except for one species which yielded no molecular data (but did exhibit morphological differences), both molecular and morphological datasets were congruent in delimiting species boundaries. Moreover, comparing the mite phylogeny with the known ecology and phylogenetic relationships of their host species suggests that these mites are coevolving with their silphid hosts rather than tracking ecologically similar species.

Description of Jolyelmis spangleri a new species from Churí-tepui (Chimantá Massif, Venezuela), with a description of the larva of J. spangleri and J. reitmaieri (Insecta: Coleoptera: Elmidae)

Four species have been recognized in the genus Jolyelmis Spangler &amp; Faitoute; all collected at isolated table mountains,mostly in cool stenothermal hypocrenal streams. The known distribution of the genus is confined to Venezuela, and eachspecies is here reported as endemic for the respective table mountain where it has been found. Samples from Churí-tepuiand Mount Roraima in south-eastern Venezuela contained numerous adults and larvae of Elmidae among which is a newspecies, Jolyelmis spangleri, which is here described. The new species is illustrated and keyed with all other Jolyelmis,and new distributional data for J. reitmaieriČiampor, Jr. &amp; Kodada are included. Molecular analysis of partial cytochromeoxidase subunit I (cox1) confirmed conspecific life stages for two species: J. spangleri and J. reitmaieri. The larva of eachspecies is described and illustrated, and the generic diagnosis for Jolyelmis larvae is provided. Among the known larvaeof South American Elmidae, larvae of the genus Xenelmis Hinton appear to be the most similar. Morphological similarities are discussed. Habitats of adults and larvae are briefly described and illustrated by photographs.

A new horsehair worm, Chordodes formosanus sp. n. (Nematomorpha, Gordiida) from Hierodula mantids of Taiwan and Japan with redescription of a closely related species, Chordodes japonensis

A new species of horsehair worm, Chordodes formosanus sp. n., is described and compared to a closely related species, Chordodes japonensis. Although both species possess the same six cuticular structures of areoles on the surface, the significantly longer filaments on the female crowned areoles can be used as diagnostic characters for the new species. The different taxonomic status of these two species was also confirmed after analyzing the partial cytochrome oxidase subunit I sequence, and the mantid hosts, which are respectively limited to the genus Tenodera for Chordodes japonensis and Hierodula for Chordodes formosanus sp. n. In addition, the immature stages of eggs and larvae of the new species are also described and discussed in detail.

Reconstructing the colonisation and diversification history of the endemic freshwater crab ( Seychellum alluaudi) in the granitic and volcanic Seychelles Archipelago

The endemic, monotypic freshwater crab species Seychellum alluaudi was used as a template to examine the initial colonisation and evolutionary history among the major islands in the Seychelles Archipelago. Five of the “inner” islands in the Seychelles Archipelago including Mahé, Praslin, Silhouette, La Digue and Frégate were sampled. Two partial mtDNA fragments, 16S rRNA and cytochrome oxidase subunit I (COI) was sequenced for 83 specimens of S. alluaudi. Evolutionary relationships between populations were inferred from the combined mtDNA dataset using maximum parsimony, maximum likelihood and Bayesian inferences. Analyses of molecular variance (AMOVA) were used to examine genetic variation among and within clades. A haplotype network was constructed using TCS while BEAST was employed to date the colonisation and divergence of lineages on the islands. Phylogenetic analyses of the combined mtDNA data set of 1103 base pairs retrieved a monophyletic S. alluaudi group comprised three statistically well-supported monophyletic clades. Clade one was exclusive to Silhouette; clade two included samples from Praslin sister to La Digue, while clade three comprised samples from Mahé sister to Frégate. The haplotype network corresponded to the three clades. Within Mahé, substantial phylogeographic substructure was evident. AMOVA results revealed limited genetic variation within localities with most variation occurring among localities. Divergence time estimations predated the Holocene sea level regressions and indicated a Pliocene/Pleistocene divergence between the three clades evident within S. alluaudi. The monophyly of each clade suggests that transoceanic dispersal is rare. The absence of shared haplotypes between the three clades, coupled with marked sequence divergence values suggests the presence of three allospecies within S. alluaudi.

Aphelenchoides paradalianensis n. sp. (Nematoda: Aphelenchoididae) isolated at Guangzhou, China, in packaging wood from South Korea

Aphelenchoides paradalianensis n. sp. collected in Guangzhou, China from packaging wood exported from South Korea, is described and illustrated based on morphology and molecular analyses of the ITS regions, the near-full-length small subunit rDNA gene and partial cytochrome oxidase subunit I of mitochondrial DNA. This new species has a distinctive tail terminus, possessing a short trunk with two finely rounded and smooth processes of unequal length in the female and a small ventral papilla-like mucronate structure or a short trunk with two fine processes in the male, slightly offset lip region with 6 annules, four incisures in the lateral field, short postuterine sac (extending ca. 13–20% of vulva-anus distance), medium-sized spicules (14.1–18.6 μ m), three pairs of caudal papillae. It is closest to A. dalianensis, but these two species can be distinguished from each other by spicule size and shape, male tail tip shape, postuterine sac length, c ratio and ITS sequence. Molecular analysis reveals that this species has unique ITS, 18S and mt-DNA sequences.

Genetic population structure of Tectura paleacea: implications for the mechanisms regulating population structure in patchy coastal habitats.

The seagrass limpet Tectura paleacea (Gastropoda; Patellogastropoda) belongs to a seagrass obligate lineage that has shifted from the Caribbean in the late Miocene, across the Isthmus of Panama prior to the closing of the Panamanian seaway, and then northward to its modern Baja California – Oregon distribution. To address whether larval entrainment by seagrass beds contributes to population structuring, populations were sampled at six California/Oregon localities approximately 2 degrees latitude apart during two post-settlement periods in July 2002 and June 2003. Partial cytochrome oxidase b (Cytb) sequences were obtained from 20 individuals (10 per year) from each population in order to determine the levels of population subdivision/connectivity. From the 120 individuals sequenced, there were eighty-one unique haplotypes, with the greatest haplotype diversity occurring in southern populations. The only significant genetic break detected was consistent with a peri-Point Conception (PPC) biogeographic boundary while populations north and south of Point Conception were each panmictic. The data further indicate that populations found south of the PPC biogeographic boundary originated from northern populations. This pattern of population structure suggests that seagrass patches are not entraining the larvae of T. paleacea by altering flow regimes within their environment; a process hypothesized to produce extensive genetic subdivision on fine geographic scales. In contrast to the haplotype data, morphological patterns vary significantly over very fine geographic scales that are inconsistent with the observed patterns of genetic population structure, indicating that morphological variation in T. paleacea might be attributed to differential ecophenotypic expression in response to local habitat variability throughout its distribution. These results suggest that highly localized conservation efforts may not be as effective as large-scale conservation efforts in near shore marine environments.

Phylogenetic relationships and evolution of pulmonate gastropods (Mollusca): New insights from increased taxon sampling

Phylogenetic relationships among higher clades of pulmonate gastropods are reconstructed based on a data set including one nuclear marker (complete ribosomal 18S) and two mitochondrial markers (partial ribosomal 16S and Cytochrome oxidase I) for a total of 96 species. Sequences for 66 of these species are new to science, with a special emphasis on sampling the Ellobiidae, Onchidiidae, and Veronicellidae. Important results include the monophyly of Systellommatophora (Onchidiidae and Veronicellidae) as well as the monophyly of Ellobiidae (including Trimusculus, Otina, and Smeagol). Relationships within Ellobiidae, Onchidiidae, and Veronicellidae are evaluated here for the first time using molecular data. Present results are compared with those from the recent literature, and the current knowledge of phylogenetic relationships among pulmonate gastropods is reviewed: despite many efforts, deep nodes are still uncertain. Identification uncertainties about early fossils of pulmonates are reviewed. Impacts of those phylogenetic and fossil record uncertainties on our understanding of the macro-evolutionary history of pulmonates, especially transitions between aquatic and terrestrial habitats, are discussed.

Phylogenetic relationships of some Sylviidae species based on complete mtDNA cyt b and partial COI sequence data

Members of the passerine family Sylviidae are distributed widely around the Old World and the phylogenetic relationships of many species still remain controversial. In this study, we investigated the phylogeny and relationships among 37 species of 11 genera by analyzing DNA sequences obtained from the complete mitochondrial cytochrome b (cyt b) and partial cytochrome oxidase I (COI) genes. The data were analyzed by maximum-likelihood analysis and Bayesian inference. The results demonstrate that the current genus Phylloscopus is non-monophyletic, while Seicercus is synonymized with Phylloscopus or is a group within the genus Phylloscopus. We may conclude that within Phylloscopus there are close relations between P. magnirostris and P. borealis; among P. proregulus, P. yunnanensis, P. pulcher and P. maculipennis, as well as among P. occisinensis, P. schwarzi, P. armandii, P. collybita and P. fuscatus. Monophyly of the genus Cettia could not be corroborated; it is closely related to the genera Abroscopus and Tesia. However monophyly of Acrocephalus is supported. Furthermore, the close relationships between Sylvia and Zosterops and among Prinia, Orthotomus and Cisticola are also supported.