Chaetopteryx bucari sp. n., a new species from the Chaetopteryx rugulosa group from Croatia (Insecta, Trichoptera, Limnephilidae) with molecular, taxonomic and ecological notes on the group

Abstract We describe a new autumnal caddisfly species Chaetopteryx bucari sp. n. from 8 localities in the Banovina region of Croatia. We also present molecular, taxonomic and ecological notes (emergence, sex ratio and seasonal dynamics) on the new species and discuss the distribution of Chaetopteryx species in general and the Chaetopteryx rugulosa group in particular. Based on Bayesian phylogenetic analysis Chaetopteryx rugulosa schmidi was separated from the clade containing the other subspecies of Chaetopteryx rugulosa. Thus the subspecies Chaetopteryx rugulosa schmidi is here raised to species level, Chaetopteryx schmidi, as it was described originally. We further present distribution data on rare species in the genus Chaetopteryx in Croatia.

Four years ago we started systematically collecting adults of the genus Chaetopteryx, including members of the C. rugulosa group in Croatia. This paper has 2 main objectives, first to present and describe a new species from the C. rugulosa group found in Croatia, and second to present new molecular, taxonomic, distributional, and ecological information on the C. rugulosa group.

Material and methods
Fieldwork. We collected specimens of Chaetopteryx including C. rugulosa group species in the continental (central Croatia, Banovina, Hrvatsko zagorje, Kordun, Slavonia), mountain (Gorski kotar, Lika regions) and Mediterranean (Istria and Dalmatia) regions of Croatia. Collecting methods included the use of entomological nets and handpicking specimens from walls of small buildings or wells, or from the riparian vegetation near springs and headwater streams. In one spring (Pecki spring, Banovina region) (Table 1) we installed 5 pyramid-type emergence traps in 2010 and 2011 to investigate the emergence dynamics of caddisflies ( Figure 1). This investigation is part of a multi-year study on emergence dynamics of aquatic insects in springs and other aquatic habitats in Croatia and the Dinaric karst of the Balkan Peninsula (Bosnia and Herzegovina) , Previšić et al. 2007 2011, 2012. The emergence trapping methodology was presented in detail by  and Previšić et al. (2007). In pyramid-type emergence traps caddisflies were collected in 1% formaldehyde and thereafter stored in 80% alcohol. All other collected specimens were stored directly in 80% or 96% alcohol. All specimens were deposited in the collections of the first and second authors. The holotype is deposited in the Croatian Natural History Museum in Zagreb.
The mitochondrial COI barcodes were generated at the Canadian Centre for DNA Barcoding, University of Guelph, Canada. Standard barcoding protocols for DNA extraction (Ivanova et al. 2006), PCR amplification and COI sequencing (Hajibabaei et al. 2005, de Waard et al. 2008 were used. Full-length COI-5P DNA barcodes were amplified using C_LepFolF/C_LepFolR (Folmer et al. 1994, Hajibabaei et al. 2006) and LCO1490/HCO2198 (Folmer et al. 1994) primer sets. COI barcodes and detailed specimen information can be found in the Barcode of Life Data Systems (BOLD; http://www.boldsystems.org/) (Ratnasingham and Hebert 2007) within the project "Chaetopteryx of Europe." Unpublished COI barcodes of additional Chaetopteryx outgroups were provided by Karl Kjer, Rutgers University, USA ( Table 2). The sequence of Limnephilus centralis Curtis, 1834 was taken from Malm and Johanson (2011) (Table 2).
Phylogenetic analysis. Sequences were edited manually and aligned using the program Geneious 5.4 (Drummond et al. 2011). The final alignment was 617 base pairs (bp) long. Bayesian phylogenetic analyses were performed using the Markov chain Monte Carlo method (B/MCMC) using MrBayes 3.2 (Buckley et al. 2002, Ronquist andHuelsenbeck 2003). We selected the best-fitting models of DNA substitution using Akaike information criterion (AIC) implemented in jModelTest 0.1.1 (Guindon andGascuel 2003, Posada 2008). jModelTest indicated a general time re- versible model (Rodríguez et al. 1990) with a significant proportion of invariant sites (I=0.607) and with gamma-distributed rate heterogeneity (α=1.049) (GTR+I+G). We conducted Bayesian tree construction with 6 chains, 2 independent runs and 8 million generations. Trees were sampled every 1000th generation. The first 9000 generations were discarded as burn-in. We plotted the log-likelihood scores of sample points against generation time using Tracer 1.5 (Rambaut and Drummond 2009) to ensure that stationary was achieved after the first 9000 generations by checking whether the log-likelihood values of the sample points reached a stable equilibrium plateau. We used the remaining trees with average branch lengths to create a 50% majority-rule consensus tree with the sumt option of MrBayes. Posterior probabilities (pp) were obtained for each clade, whereby pp≥0.95 indicated significant support for clades. Finally, we also calculated the uncorrected pairwise distances between individuals based on mtCOI sequences using MEGA 5.1 (Tamura et al. 2011). Microphotography and measuring. Microphotographic images of genitalia and forewing measurements were taken using a Leica Wild MZ8 stereomicroscope and Olympus SP-500 UZ digital camera. The photographs were processed with the Olympus Quick Photo Camera 2.2. software package. Geographic coordinates and altitudes of sampling localities were recorded with a Garmin 'Oregon 450' GPS device.

Phylogenetic analyses.
In the Bayesian phylogenetic tree based on mtCOI sequences the C. rugulosa group species clustered into 4 strongly supported clades (Figure 2). Chaetopteryx marinkovicae was basal within the species group. The remaining species fell into 3 clades: a basal clade with C. r. schmidi, C. bucari sp. n., and 2 derived sister clades comprising C. clara, C. goricensis, C. irenae, and C. r. rugulosa, C. r. noricum, C. r. mecsekensis. Chaetopteryx bucari sp. n. is sister to the highly supported C. r. schmidi. The mean value of the uncorrected pairwise distance (p distance) was 2.02% between them (Table 3). The p distance did not reach 1% within the 2 clades (C. bucari sp. n.: 0.17%; C. r. schmidi: 0.75%). The relationship of the nominal species of the group C. r. rugulosa and C. r. noricum was not resolved, as the 4 subclades formed a polytomy. In the phylogenetic tree C. r. schmidi was clearly separated from the clade containing the subspecies of C. rugulosa (Figure 2). The mean values of p distance between the 3 subspecies of C. rugulosa ranged between 1.61-3.02 %, while the mean values between the C. r. schmidi and the other subspecies of C. rugulosa were distinctly higher (4.66 -5.85%) ( Table 3). Diagnosis. Male of C. bucari is most similar to C. r. mecsekensis and C. r. schmidi but differs in the following features: 1. In lateral view the inferior appendages in C. bucari are always with a pointed apex on the dorsal side, not rounded as in C. r. mecsekensis; 2. Bristles in C. bucari are set more distally from the membranous part of the aedeagus than in C. r. mecsekensis and C. r. schmidi and never reach (touch) the lateral Table 3. Estimates of evolutionary divergence over sequence pairs within and between phylogenetically defined species and subspecies based on mtCOI sequence data. Distance matrix is shown with the mean ± SD values of the intraspecific and interspecific pairwise genetic distances for the all Chaetopterygini species included in the analysis. Abbrev.: CRR -Chaetopteryx rugulosa rugulosa, CRN -C. r. noricum, CRM -C. r. mecsekensis, CCX -C. clara, CGR -C. goricensis, CIX -C. irenae, CBU -C. bucari sp.n., CRS -C. schmidi, CMR -C. marinkovicae, CBO -C. bosniaca, CMO -C. morettii, CFU -C. fusca, CVI -C. villosa, CGE -C. gessneri, CAX -C. aproka, CMA -Chaetopterygopsis maclachlani, CMJ -Chaetopteryx major. CRM 11.89±0.07 11.97±0.08 11.76±0.14 11.76±0.14 12.44±0.14 11.60±0.12 11.47±0.08 13.78±0.00     membranous finger, as in C. r. mecsekensis. Female of C. bucari is clearly different from other species in the C. rugulosa group (e.g., form of the visible finger on lateral side, form of the anal tube, form of the supragenital plate of segment X in lateral and ventral views, form of the median lobe of the vulvar scale in ventral view). We did not find strong morphological variability among the females of the new species (except the median lobe of the vulvar scale). Females of C. bucari have in lateral, ventral and dorsal views very visible finger-shaped proturbances (ventral lobes of tergite IX) on the anal tube which is lacking in C. r. mecsekensis and C. r. schmidi. In lateral view the excision of the anal tube in C. r. rugulosa is more pronounced than in C. bucari. The median lobe of the vulvar scale in C. r. mecsekensis, C. r. rugulosa and C. r. schmidi is longer and more visible than in C. bucari. Description. Wings and legs yellow to yellowish-brown; veins darker in both sexes (Figure 3). Antennae long, grey to fuscous. Scapus yellow to yellowish-brown, thorax and abdomen yellow. Spur formula male 0,3,3, female 1,3,3. Ocelli present. Forewing with round apex; length 7.7-9.9 mm in males, 7.2-10.1 mm in females.
Female genitalia (Figures 12-16). Anal tube (fusion of tergites IX and X) in lateral view broad, relatively elongated with one excision and very distinct finger-shaped proturbance (lobes of tergite IX) on ventral side (Figures 12-13). Apex of proturbance rounded or slightly pointed with small yellow setae (Figures 12-15). In 2/3rds of specimens examined ventral and dorsal lips of anal tube equal in length, in 1/3rd ventral lip longer. In dorsal view anal tube thickened with digitate proturbance on lateral side    and small excision (recess) in middle ( Figure 14). In ventral view anal tube broad with larger excision (recess) in middle than in dorsal side ( Figure 15). Supragenital plate of segment X well-developed, triangular in shape in lateral and ventral views (Figures 12,  15). Lateral segment of vulvar scale relatively short in ventral view, with flat or slightly rounded apex (Figure 16a-c). Median lobe of vulvar scale (lower vulvar lip) with very small rounded or pointed apex (Figure 16b-d). In ca. 1/3rd of specimens' median lobe of vulvar scale not visible (Figure 16a).
Etymology. The species is dedicated to Professor Matija Bučar from the Faculty of Education, Department in Petrinja, University of Zagreb.     nja and Gore). We collected C. bucari from 2 springs, 5 wellsprings and 1 location in the stream (Table 1). In total, we collected more than 580 specimens of C. bucari (85% were collected in pyramid-type emergence traps). The most abundant populations were found at Pecki spring and a headwater stream in Hrvatski Ćuntić. Over 150 specimens of C. bucari were observed on the night of October 14, 2010 on the walls of a small building next to the stream in Hrvatski Čuntić. In Pecki spring more than 50 specimens were observed on the night of October 31, 2010. Chaetopteryx bucari was recorded at low altitudes between 104-185 m a.s.l. (Table 1).
In addition to C. bucari 2 other species of the C. rugulosa group were collected in Croatia during our recent surveys. Chaetopteryx marinkovicae was collected from its type locality on the stream and spring in Kompanj village (Istria region); C. r. rugulosa was caught on Mt. Žumberak and Mt. Medvednica (northeast and central Croatia). Other species of Chaetopteryx found during this investigation were Chaetopteryx bosniaca Marinković-Gospodnetić, 1959 (Lika region), Chaetopteryx gonospina Marinković-Gospodnetić, 1966 (Banovina region), C. fusca (central Croatia, Dalmatia and Lika regions), and C. major (central Croatia).

Discussion
Systematic and taxonomic implications. Based on molecular evidence, we could confirm the hypothesis that Chaetopteryx bucari is a distinct species. Although C. bucari does not have a pp >0.95, it represents the sister taxon (pp>0.95) to the highly supported C. r. schmidi. Furthermore, the mean genetic distance (2.02%) between C. bucari and C. r. schmidi barely reached the 2-3% divergence observed as an interspecific genetic divergence in mtCOI sequences among some well-defined caddisfly species (Bálint et al. 2009, Pauls et al. 2009. However, among other well-defined caddisfly species this value can reach much higher levels (e.g., Zhou et al. 2007, Pauls et al. 2010), but also much lower values (e.g., Waringer et al. 2007). Thus reliance on distance methods alone for defining species boundaries is not advisable and species boundaries should be supported by additional lines of evidence such as additional, independent genes, morphology, or other independent characteristics (Zhou et al. 2007), particularly in taxa where hybridization is possible as is the case in Chaetopteryx Pauls 2012). In the present study the genetic distinctiveness of C. bucari in combination with differences in morphological characters compared to its congeners, provide strong evidence to justify describing it as a new species.
In both sexes, especially in the adult female, C. bucari is relatively easily distinguishable from other taxa of the C. rugulosa group. The genetic data also show that specimens from 7 populations across the known range of the species from a clearly distinct clade from all other analysed Chaetopteryx. It is interesting that the female of C. bucari is particularly informative in diagnosing the species. In caddisflies this is quite unusual as males are generally more easily distinguished and females are often very difficult to differentiate from one another.
Based on the phylogenetic position of C. r. schmidi in relation to C. r. rugulosa and the other C. rugulosa subspecies, C. r. schmidi is well-defined and quite divergent from other members of the C. rugulosa clade based on molecular data. Thus, the subspecies C. r. schmidi is here re-established as a distinct species, C. schmidi, as it was described According to the current findings, C. bucari is not rare in the Croatian fauna. In fact, it is one of the most dominant caddisflies in the Banovina region. Along with C. fusca , Semnički et al. 2011, Cerjanec 2012 it is one of the most frequently found species from genus Chaetopteryx in Croatia. C. bucari inhabits springs and headwaters of small streams. The only known larger limnocrene spring that C. bucari inhabits is the Pašino vrelo spring.
Taxa from the C. rugulosa group have allopatric distributions in Croatia ( Figure  17): C. bucari is distributed in the Banovina region, C. r. rugulosa in northern Croatia on Mt. Medvednica and Mt. Žumberak, C. r. mecsekensis in eastern Croatia on Mt. Papuk and C. marinkovicae in the sub-Mediterranean part of Croatia in Istria (Malicky and Krušnik 1988. Systematic research in mountain areas in Lika and Gorski kotar , Previšić and Popijač 2010, Cerjanec 2012, Semnički et al. 2011, 2012 and the Mediterranean part of Croatia (Dalmatia region) (Graf et al. 2008, Waringer et al. 2009, Vučković 2011 did not result in collections of C. rugulosa group species in these areas.
Many members of the genus Chaetopteryx are either small-scale endemics or species with a low number of disjunct populations. This makes the group very interesting for biogeographic studies. There are several reasons that could explain the observed pattern of distribution: small populations, poor mobility of the winter emerging adults, and distribution in springs and in headwater reaches of small streams. Besides naturally isolating individual populations from one another, these aspects can also cause difficulties for investigating the genus, as it is hard to access many of the sites, especially in winter. Future investigations of this genus will be focused on poorly researched areas in Croatia and the western Balkans to gain a better understanding of the distribution and biogeography of Chaetopteryx in the region.