Ecological comparison between duckweeds in central Italy: The invasive Lemna minuta vs the native L. minor

The American duckweed Lemna minuta shows an invasive behaviour in Europe, causing weed problems in aquatic habitats there. Few studies addressed this species’ ecological requirements for a suitable establishment in a site. In this paper, L. minuta populations were analysed through field surveys so as (1) to define the autoecology of this duckweed as regards the main environmental factors characterizing invaded habitats, and (2) to identify possible overlaps/differences in ecological requirements between the alien L. minuta and the common native L. minor, with which it is often associated and in direct competition. The occurrence/abundance of the two species and environmental data were collected from 41 wetlands in central Italy. Currently, L. minuta is more common and abundant than L. minor in the study-area, despite its recent arrival there (2007). The two species have a partially overlapped autoecology. However, L. minuta differs from L. minor since it occurs in waters which are less alkaline, slightly less warm, and richer in nitrates. It shows tolerance for environmental conditions which are limiting for most of macrophytes, including L. minor, such as high shading and low water oxygenation. This enables L. minuta to increase its invasion potentiality and thus to enlarge its distribution area.     

Genetic structure of the genus Lemna L. (Lemnaceae) as revealed by amplified fragment length polymorphism

Duckweeds (Lemnaceae) are extremely reduced in morphology, which made their taxonomy a challenge for a long time. The amplified fragment length polymorphism (AFLP) marker technique was applied to solve this problem. 84 clones of the genus Lemna were investigated representing all 13 accepted Lemna species. By neighbour-joining (NJ) analysis, 10 out of these 13 species were clearly recognized: L. minor, L. obscura, L. turionifera, L. japonica, L. disperma, L. aequinoctialis, L. perpusilla, L. trisulca, L. tenera, and L. minuta. However, L. valdiviana and L. yungensis could be distinguished neither by NJ cluster analysis nor by structure analysis. Moreover, the 16 analysed clones of L. gibba were assembled into four genetically differentiated groups. Only one of these groups, which includes the standard clones 7107 (G1) and 7741 (G3), represents obviously the “true” L. gibba. At least four of the clones investigated, so far considered as L. gibba (clones 8655a, 9481, 9436b, and Tra05-L), represent evidently close relatives to L. turionifera but do not form turions under any of the conditions tested. Another group of clones (6745, 6751, and 7922) corresponds to putative hybrids and may be identical with L. parodiana, a species not accepted until now because of the difficulties of delineation on morphology alone. In conclusion, AFLP analysis offers a solid base for the identification of Lemna clones, which is particularly important in view of Lemnaceae application in biomonitoring.     

Non-native fish species in Neotropical freshwaters: how did they arrive,and where did they come from?

The invasion of aquatic ecosystems by introduced invasive alien species (IAS) has become a worldwide phenomenon, and often leads to competitive interactions with native species. At high-nutrient levels, native species mostly are outcompeted by the introduced species. We performed an outdoor competition experiment between IAS free-floating Lemna minuta and native Lemna minor in a eutrophicated pond to examine whether the invasive species is the better competitor. We additionally performed an indoor experiment resembling mesotrophic phosphorus (P) conditions to investigate both species’ competitiveness in low P availability and compared with previous experiments at high-nutrient levels. Our results showed that in field conditions, the alien L. minuta was the better competitor. In the mesotrophic indoor condition, however, the native L. minor was the better competitor. Both species produced longer roots in the indoor experiment compared to field conditions. The species’ relative growth rates were also lower in the indoor experiment. A P reduction to mesotrophic condition in the water column thus might reduce invasive L. minuta growth and competitive performance. Additionally, introduction and recovery of L. minor could reduce L. minuta cover, but only following P reduction. Field experiments in mesotrophic ponds are needed to confirm these indoor findings.     

DNA barcoding approach fails to discriminate Central European bladderworts (Utricularia,Lentibulariaceae), but provides insights concerning their evolution

Abstract

The main features to distinguish the seven native Utricularia species occurring in central Europe are found in flower shape, but being rarely flowering identification is often doubtful and uncertain. A recent morphometric work highlighted that there are no univocal reliable extra-floral morphological features allowing a safe identification at species level. Therefore, DNA barcoding approach is attempted here. Molecular analyses were performed to search for DNA barcodes using nuclear ITS (rDNA), plastid (cpDNA) trnL-trnF IGS and rps16 intron sequences. Generally, the barcoding approach failed to discriminate Utricularia species, although it could be of some help in the U. minor aggregate. With few exceptions, U. bremii shows peculiar DNA regions different from U. minor for both plastid markers investigated. However, interesting hypotheses could be derived from the obtained networks, including hybridization events to explain the rise of mostly sterile species, such as U. stygia. This species clusters with the other species of the U. intermedia aggregate in plastid phylogenetic graphs, while it is closely related to species of the U. minor aggregate in ITS phylogenetic graphs. Additionally to U. stygia, U. ochroleuca also shows some incongruences in the different markers, at least for some accessions, pointing to the possible occurrence of hybrids.     

Low levels of intraspecific genetic variation at a rapidly evolving chloroplast dna locus in North American duckweeds (Lemnaceae)

Although most previous studies on chloroplast (cp) DNA variation in plants have concentrated on systematics and evolution above the species level, intraspecific variation in cpDNA is common and has provided useful insights into population-level evolutionary processes. Polymerase chain reaction methods were used to examine restriction site and sequence variation in the chloroplast rpLI6 gene within and among populations of duckweed species (Spirodela and Lemna) from the southern and eastern United States. To our knowledge, the rpL16 region has not previously been used to investigate cpDNA variation in nature. While considerable restriction site and sequence variation were detected among species, no variation was found within populations of either of the two species (S. punctata and L. minor) selected for sequence analysis, and S. punctata showed no interpopulational variation. Two cpDNA haplotypes were identified in L. minor, with one haplotype restricted to three sites in Louisiana and the other found in all other populations sampled. This paucity of variation cannot be readily explained as the result of a low mutation rate. In general, group II introns appear to be subject to very little functional constraint, and extensive sequence differences have been found between species in the chloroplast rpL16 intron in particular. However, factors such as historical range expansions and contractions, founding effects, fluctuations in local population size, and natural selection may play a role in reducing cpDNA sequence variability in these species.     

DNA barcoding of the South Korean Tettigoniidae (Orthoptera) using collection specimens reveals three potential species complexes

We carried out DNA barcoding on 24 Korean tettigonid species of 19 genera deposited in the National Institute of Biological Resources to reevaluate the preliminary identification of each specimen. Sequence divergence of DNA barcodes obtained from 113 samples of the 24 species ranged from 0 to 30.4%, the intraspecific variation was 0–7.3%, and the interspecific divergence was 1.1–30.4%; we could not examine the barcoding gap. In the neighbor‐joining tree, the branch length among individuals of Tettigonia ussuriana, Paratlanticus ussuriensis, and Hexacentrus japonicus were relatively longer than those in other species. The detailed analysis of the morphological characters and DNA barcodes of the above three species revealed that these three species represent species complexes. The T. ussuriana complex comprised T. jungi, T. uvarovi, and T. ussuriana. Paratlanticus ussuriensis cluster contained four species; one cluster was identified as P. palgongensis based on morphological characteristics, but the other three clusters, including the P. ussuriensis cluster, require further detailed taxonomic analysis. Lastly, two species clusters were identified within the Hexacentrus japonicus clade. Based on the 99% sequence similarity obtained by blast search of the NCBI GenBank database, one of the clusters was identified as H. unicolor. Thus, the DNA barcoding revealed the presence of at least three cryptic species in Korean Tettigoniidae, although more detailed taxonomic analyses are required to establish their status. Therefore, we suggest that DNA barcoding is a very useful tool for increasing the identification accuracy of insect collections.     

Revision of the systematics of the genus Calliptamus Serville 1831, (Orthoptera: Acrididae: Calliptaminae) in Algeria using morphological,chemical, and genetic data

Summary. The genus Calliptamus contains swarming orthopterans that cause serious damage in Algerian agricultural systems. However, it remains difficult to identify species within this genus; a thorough understanding of the group’s systematics and the utilization of novel taxonomic criteria are needed. We used morphological analysis along with two other methods of species identification – chemotaxonomy with cuticular compounds and DNA barcoding involving the COI gene – to classify 81 individual grasshoppers collected at two different sites in the Sétif region (northeastern Algeria). The chemotaxonomic analyses yielded ambiguous results, but DNA barcoding allowed us to differentiate two Calliptamus species found in Algeria: Calliptamus barbarus (Costa 1836), and Calliptamus wattenwylianus (Pantel 1896). Several morphological criteria used in identification keys appear to reflect differences among morphotypes rather than differences between species, and their taxonomic specificity is not supported by the barcoding data. The number of spines on the hind tibia is the only morphological criterion that reflected genetic differences between species; it is thus considered to be a taxonomically useful feature for identifying species in this genus.     

Integrative taxonomy at work: DNA barcoding of taeniids harboured by wild and domestic cats

In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free‐ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post‐mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already‐described taeniid species. Indeed, we detected three well‐defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.     

莪术基原植物DNA条形码序列的筛选与鉴定

为寻找适用于中药材莪术基原植物鉴定的DNA条形码序列,探索快速高效的莪术基原植物鉴定的新方法,该文首先利用扩增成功率和测序成功率对中药材莪术三种基原植物,9个样本的7种DNA条形码序列(ITS、ITS2、matK、psbA-trnH、trnL-trnF、rpoB和atpB-rbcL)进行评估,然后利用MEGA6.0软件对获得的高质量的序列通过变异位点分析、遗传距离计算和系统树分析等进一步进行评估,最后将筛选到的DNA条形码序列对未知基原的待测样品进行基原鉴定。结果表明:(1) ITS、ITS2和matK等条形码序列在莪术基原植物中的扩增或测序成功率较低,难以应用于实际鉴定;而psbA-trnH、trnL-trnF和rpoB条形码序列变异位点信息过少,不足于区分莪术的三种不同基原植物;只有atpB-rbcL条形码序列的扩增和测序成功率较高,容易获得高质量的序列,同时序列长度(642～645 bp)理想,变异位点多(11个),可实现莪术的三种不同基原的区分鉴别。(2)待测样品经基于atpB-rbcL序列构建的系统发育树鉴别为温郁金。综上所述,叶绿体atpB-rbcL序列能够准确鉴定莪术不同基原植物,可以作为中药材莪术基原植物鉴定的条形码序列。     

Competition between Lemna minuta and Lemna minor at different nutrient concentrations

We investigated the differential responses of invasive alien Lemna minuta and native Lemna minor to nutrient loading as well as the mechanism of competition between the species. The role of nutrients, species identity, species influence in determining the outcome of competition between the species was estimated using the Relative Growth Rate Difference (RGRD) model. The two species differed in their response to nutrient loading. The native L. minor responded indifferently to nutrient loading. The species Relative Growth Rate (RGR) was 0.10 d⁻¹, 0.11 d⁻¹ and 0.09 d⁻¹ in high, medium and low nutrients, respectively. On the other hand, the invasive L. minuta responded opportunistically to high nutrient availability and had an RGR of 0.13 d⁻¹, 0.10 d⁻¹ and 0.08 d⁻¹ in high, medium and low nutrients, respectively. As a result, the invasive species was dominant in high nutrient availability but lost to the native species at low nutrient availability. The invader formed approximately 60% and less than 50% of the stand final total dry biomass in high and low nutrient availability, respectively. Species RGR were reduced by both intra- and interspecific competition but intraspecific effects were stronger than interspecific effects. On the overall, the species significantly differed in their constant RGR. These differences in RGR between the species (species identity) and the differential response to nutrient loading were the main determinant of change in final biomass composition of these species in mixture. Species influence (competition) only had a small influence on the outcome of competition between the species. The observed species response to nutrient loading could be targeted in management of the invasive species. Lowering nutrients can be proposed to reduce the impact of the invasive L. minuta.     

The new threat to Italian inland waters from the alien crayfish “gang”: the Australian Cherax destructor Clark, 1936

Biological invasions inflict damage to the ecology, economy, and human health, and pose serious threats to the native communities. Among the many invasive taxa, crayfish have attracted much attention by scientists and policy makers. Recently, an established population of an alien species of crayfish, new for Italy, was found in the Natural Preserve of “Laghi di Ninfa” (central Italy). Based on morphological and genetic evidence (molecular barcoding of COI and 16S rDNA), we classified it at subspecies level as the Parastacidae Cherax destructor destructor, native to Australia. Its introduction possibly occurred at the end of the 1980s but the species seems to be still confined in the preserve. The low temperature of the adjacent waters may be a barrier against its natural spreading but not against its intentional translocation into natural waters by man. Because of the invasive history of C. destructor, eradication of this population is urgent and still economically profitable. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: Pierluigi Viaroli     

DNA barcoding reveals a cryptic nemertean invasion in Atlantic and Mediterranean waters

For several groups, like nemerteans, morphology-based identification is a hard discipline, but DNA barcoding may help non-experts in the identification process. In this study, DNA barcoding is used to reveal the cryptic invasion of Pacific Cephalothrix cf. simula into Atlantic and Mediterranean coasts. Although DNA barcoding is a promising method for the identification of Nemertea, only 6 % of the known number of nemertean species is currently associated with a correct DNA barcode. Therefore, additional morphological and molecular studies are necessary to advance the utility of DNA barcoding in the characterisation of possible nemertean alien invasions.     

DNA barcoding as an effective tool to complement wetland management: A case study of a protected area in Italy

In recent years, DNA barcoding has been suggested as a useful molecular technique to complement traditional taxonomic expertise for fast species identification and biodiversity inventories. In this study, in situ application of DNA barcodes was tested on the plant community of a wetland area in central Italy. Four cpDNA markers (trnH–psbA, rbcL, rpoC1, and matK) were tested on 40 plant species, 26 of which strictly connected to the aquatic habitat. Universality of the method, ease of data retrieval, and correct assignation of the genetic markers to each species were evaluated. The markers showed different prospects of reliable applicability. The obtained sequences were blasted against the NCBI database to verify the correct species identification. A score ranging between 32% and 67% was achieved. Overall, eight species remained unidentified with all the tested barcodes due to the absence of conspecific sequences in the available databases. This work demonstrates some limitations in the applicability of DNA barcoding to accomplish complete taxonomical surveys. Difficulties encountered in this study urge refinement of technical protocols and accessibility to wider databases. Future technological advances and larger sample sets will certainly reinforce DNA barcoding as a useful tool to address knowledge and conservation of wetlands.     

THE DISTRIBUTION AND TAXONOMIC SIGNIFICANCE OF LIGNIN IN THE LEMNACEAE

Lignification characteristics of 14 species of Lemnaceae were studied by the alkaline oxidation technique in an attempt to resolve conflicting reports of the presence of lignin and to provide chemotaxonomic data for supplementing previous systematic treatments. Four species of Spirodela, eight of Lemna, Wolffiella oblonga, and Wolffia microscopica were clonally subcultured, homogenized, exhaustively extracted and the insoluble residue oxidized in alkaline cupric hydroxide. Barley (Hordeum vulgare), Elodea densa and Spirogyra sp. were examined to validate the technique. The benzaldehydes were extracted, separated by thin-layer chromatography, and quantitated spectrophotometrically. Recovery of syringaldehyde, vanillin, and to a lesser extent p-hydroxybenzaldehyde, was accepted as evidence that these plants are lignified. The highest yields of syringaldehyde and vanillin were recovered from S. intermedia, the largest member of the Lemnaceae. Lesser amounts were obtained from S. polyrhiza and S. oligorhiza. Spirodela biperforata yielded no syringaldehyde which substantiates its distinctiveness from the morphologically similar S. polyrhiza. All Lemna species are nerved, yet vanillin (but not syringaldehyde) was recovered only from L. trinervis, L. perpusilla, L. minor, and L. minima. The species L. trisulca, L. gibba, L. obscura, and L. valdiviano yielded only p-hydroxybenzaldehyde. Thus the presence of nerves is not evidence for lignification within the genus Lemna, and the relationships suggested by their gross morphology are not supported by their lignin chemistry. Wolffiella is non-lignified by these criteria. Wolffia microscopica is the smallest flowernig plant and it has no nerves; vanillin was unexpectedly recovered and microscopic examination showed highly lignified anther walls.     

A DNA Barcoding Approach to Characterize Pollen Collected by Honeybees

In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno), characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella) at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands.     

Molecular detection of Leishmania (Sauroleishmania) tarentolae in human blood and Leishmania (Leishmania) infantum in Sergentomyia minuta: unexpected host-parasite contacts

The detection of atypical Kinetoplastida in vertebrate hosts and vectors might suggest unexpected host-parasite contacts. Aside to major vectors of Leishmania (Leishmania) infantum in Italy (e.g. Phlebotomus perniciosus and Phlebotomus perfiliewi), the sand fly fauna also includes Sergentomyia minuta, herpetophilic and proven vector of Leishmania (Sauroleishmania) tarentolae, in which records of blood meal on mammals and detection of L. infantum DNA are increasing. This study was conducted in Central Italy aiming to molecularly detect potential atypical Leishmania host-vector contacts. Detection of Leishmania spp. DNA was performed by polymerase chain reaction (SSU rRNA, ITS1 targets) on field-collected sand fly females (N = 344), blood samples from humans (N = 185) and dogs (N = 125). Blood meal identification was also performed on engorged sand flies. Leishmania spp. DNA was found in 13.1% sand flies, 3.7% humans and 14.4% dogs. Sequence analysis identified L. infantum in S. minuta (4.4%), P. perniciosus (9.1%), humans (2.2%) and dogs (14.4%). Leishmania tarentolae was detected in S. minuta (12.6%), P. perfiliewi (6.6%) and human (1.6%) samples. Of 28 S. minuta examined for blood meal, 3.6 and 21.4% scored positive for human and lizard DNA, respectively. These results indicate the importance of one-health approach to explore new potential routes of transmission of leishmaniasis involving S. minuta.     

Don't judge a fish by its fins: species delineation of Congolese Labeo (Cyprinidae)

Conspicuous characters are often useful in species identification. Yet, identification and delineation are two different processes, and such characters do not necessarily provide the best basis on which species can be delineated. This is illustrated by the case of the Labeo with papillary lips from the Congo basin. Traditionally, species delineation in this group was based on a conspicuous trait: the shape of the dorsal fin, which shows a profound degree of differentiation. Morphometric analyses were performed on 185 specimens both with and without measurements taken on this fin. The groups obtained using these two approaches were compared with those obtained through DNA barcoding. For this, 24 sequences of the standard barcoding COI gene were obtained. Species delineations based on morphological and molecular results were in agreement when the shape of the dorsal fin was ignored. This suggested that of the five nominal species known from the Congo basin, L. altivelis, L. rosae, L. lineatus, L. weeksii and L. maleboensis, only the former three remain valid. Consequently, L. weeksii was synonymised with L. altivelis and L. maleboensis with L. lineatus. The sole Congo basin endemic is L. lineatus as L. altivelis and L. rosae also occur in more southern basins. The use of the shape of the dorsal fin in morphological studies has previously led to overestimates of species diversity in this group. This is due to the fact that L. altivelis shows a remarkable amount of geographical variation for this trait. The large amount of intra‐ and interspecific variation in this character was caused by differential allometric growth in different parts of the dorsal fin.     

Diadromus pulchellus in North America: field release against leek moth and new characters to distinguish it from Diadromus subtilicornis,a native diamondback moth parasitoid

We report successful overwintering of Diadromus pulchellus in North America (Ontario) following introduction of this species from Europe to control the leek moth, Acrolepiopsis assectella, a recently established alien species. Field rearing revealed that the native Diadromus subtilicornis emerged only from diamondback moth, Plutella xylostella, whereas D. pulchellus was reared almost exclusively from leek moth. The single D. pulchellus reared from diamondback moth was anticipated because host range studies found this species could develop on both leek moth and diamondback moth in the laboratory, although, it had not been previously reported from diamondback moth in the field in Europe. DNA barcoding of specimens of both Diadromus spp. confirmed their species status and novel morphological characters are presented to distinguish D. pulchellus from D. subtilicornis. In addition, DNA from specimens of D. subtilicornis from Europe clustered with DNA from specimens across Canada, confirming that it is a single Holarctic species. Finally, a new host association for D. subtilicornis is recorded from the dame's rocket moth: Pseudoplutella porrectella.     

Allozymic and morphometric variation inLemna minor (Lemnaceae)

Allozymic and morphometric variation was studied in 28 clones ofLemna minor. This variation was compared with the corresponding variation in four clones ofLemna gibba and four clones ofSpirodela polyrrhiza. A high level of allozymic variation was observed among the clones, despite having been grown under uniform laboratory conditions for several years and despite its quasi-exclusive clonal means of propagation. Based on degree of allozymic similarity,Spirodela polyrrhiza was distinguished from the twoLemna species but the latter species were genetically indistinguishable. Allozymic similarity among clones ofLemna minor was not related to morphometric similarity, nor was it related to the degree of geographic separation or climatic similarity of their sites of origin. The results suggest that allozymic variation among these clones ofLemna minor may be largely neutral and not a consequence of differential selection.     

“Forms” of water mites (Acari: Hydrachnidia): intraspecific variation or valid species?

In many groups of organisms, especially in the older literature, it has been common practice to recognize sympatrically occurring phenotypic variants of a species as “forms”. However, what these forms really represent often remains unclear, especially in poorly studied groups. With new algorithms for DNA‐based species delimitation, the status of forms can be explicitly tested with molecular data. In this study, we test a number of what is now recognized as valid species of water mites (Hydrachnidia), but have in the past been treated as forms sympatrically occurring with their nominate species. We also test a form without prior taxonomical status, using DNA and morphometrics. The barcoding fragment of COI, nuclear 28S and quantitative analyses of morphological data were used to test whether these taxa merit species status, as suggested by several taxonomists. Our results confirm valid species. Genetic distances between the form and nominate species (Piona dispersa and Piona variabilis, COI 11%), as well as likelihood ratio tests under the general mixed‐Yule coalescent model, supported that these are separately evolving lineages as defined by the unified species concept. In addition, they can be diagnosed with morphological characters. The study also reveals that some taxa genetically represent more than one species. We propose that P. dispersa are recognized as valid taxa at the species level. Unionicola minor (which may consist of several species), Piona stjordalensis, P. imminuta s. lat., and P. rotundoides are confirmed as species using this model. The results also imply that future studies of other water mite species complexes are likely to reveal many more genetically and morphologically distinct species.