Two new species of Strigula (lichenised Dothideomycetes, Ascomycota) from China, with a key to the Chinese foliicolous species

Strigula has traditionally been circumscribed based on morphology, but species delimitation in the genus generally lacks comprehensive analyses. A molecular approach has now been applied to foliicolous material of the genus from tropical areas in China. On the basis of combined phenotyic and genotypic data, two new species are described from southern China: S. acuticonidiarum and S. guangxiensis.

During our studies of the lichens of China, two species of Strigula new to science have been found as a result of integrated phenotypic and molecular analyses.

Phenotypic analyses
All specimens of Strigula examined in this study were collected by the first author from Guangxi Province in China and are preserved in the Fungarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS-L). A Leica M125 dissecting microscope was used for the morphological studies and a Zeiss Axioscope2 compound microscope with a Zeiss Axio Imager A2 was used for the anatomical studies. Sections were studied and photographed with an AxioCam MRc5 camera in tap water. Thin-layer chromatography (TLC) (Culberson and Kristinsson 1970, Culberson 1972, Orange et al. 2001 was applied for the detection of lichen substances.

Genotypic analyses
Eighteen fresh specimens of Strigula were chosen for DNA extraction (Table 1), for which a modified CTAB method (Rogers and Bendich 1988) was used. Primers ITS5 and ITS4 were used to amplify the nrRNA gene ITS region (White et al. 1990). Reactions were carried out in a 25 µl reaction volume including 1 µl DNA, 1 µl each primer (10 µM), 2 µl dNTP (2.5 mM), 2.5 µl amplification buffer (containing 25 mM Mg 2+ ), 0.5 µl Taq polymerase and 17 µl ddH 2 O. Cycling parameters were set to an initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 50 s and a final extension at 72°C for 10 min. The new sequences generated for this study are deposited in GenBank (Table 1).
Twenty-seven sequences were aligned with the program MAFFT (Katoh 2002), including 18 newly generated for this study (Table 1). Seven sequences were taken
Chemistry. No substances detected by TLC.
Habitat and distribution. On the surface of living leaves in humid, semi-exposed forests of south China.
Etymology. The epithet "acuticonidiarum" is a compound of a Latin adjective "acutatus" (a, um, and acuti-in Latin comp.) meaning sharply pointed, and "macroconidiarum", a plural genitive of the Latin neuter noun, "macroconidium". This recalls the acute ends of the macroconidia.
Other specimens examined. CHINA. Guangxi: Nanning City, Long'an County, Longhu mountain natural reserve. Remarks. Strigula antillarum can be distinguished from the new species by the perithecia being immersed only at base, aggregate and confluent pycnidia forming black spots or radiating lines, and bacillar, shorter, macroconidia (12-20 × 3-4 µm) with rounded ends (Lücking 2008). The new species is externally most similar to S. smaragdula (Figure 2e-i), in which perithecia are covered by the bright green thallus (Santesson 1952, Lücking 2008. However, S. acuticonidiarum is characterized by a thinner thallus with entire to crenulate margins (thallus 20-80 µm thick in S. smaragdula), the absence of distinct lobes, and in having small and round thalli instead. Anatomically, it differs in the shorter asci and the macroconidia having more acute ends. In molecular analyses, the ITS rDNA sequences confirmed it as different from S. smaragdula ( Figure  1). The two species are distinct both morphologically and phylogenetically.
Strigula smaragdula is generally considered to be a very common but variable species, traditionally recognized morphologically, for example by the thallus having entire to crenulate or lobulate margins, and sometimes the whole thallus being lobate-lacin-iate. This variation series has been regarded as merely due to environmental or habitat modification. However, the most common state, represented by the holotype of S. smaragdula, is characterised by distinct, but short and rounded marginal lobes (Santesson 1952). The broad concept of S. smaragdula evidently represents a species complex, rather than a single species. Minor morphological traits, including thallus form and differences in ascus size and the shape of macroconidia, are diagnostic for segregating S. acuticonidiarum from S. smaragdula s. str., a distinction supported by molecular data (Figure 1). Diagnosis. Characterized by the thin thallus (30-45 µm thick), long asci (45-65 × 10-12.5 µm), aggregated pycnidia, large ascospores (15-25 × 2.5-5 µm), and 1-septate macroconidia (12. 5-17.5 × 2.5-5 µm).
Chemistry. No substances detected by TLC. Habitat and distribution. On the surface of living leaves in humid, semi-exposed forests of south China.
Etymology. The epithet "guangxiensis" is the name of the province including the type locality of the new species.