Nozaki et al. Bot Stud (2018) 59:10 https://doi.org/10.1186/s40529-018-0227-9 ORIGINAL ARTICLE Open Access Morphology, taxonomy and mating‑type loci in natural populations of Volvox carteri in Taiwan Hisayoshi Nozaki1* , Noriko Ueki2, Mari Takusagawa3, Shota Yamashita1, Osami Misumi4, Ryo Matsuzaki5, Masanobu Kawachi5, Yin‑Ru Chiang6 and Jiunn‑Tzong Wu6 Abstract Background: Volvox carteri f. nagariensis is a model taxon that has been studied extensively at the cellular and molecular level. The most distinctive morphological attribute of V. carteri f. nagariensis within V. carteri is the produc‑ tion of sexual male spheroids with only a 1:1 ratio of somatic cells to sperm packets or androgonidia (sperm packet initials). However, the morphology of male spheroids of V. carteri f. nagariensis has been examined only in Japanese strains. In addition, V. carteri f. nagariensis has heterothallic sexuality; male and female sexes are determined by the sex-determining chromosomal region or mating-type locus composed of a > 1 Mbp linear chromosome. Fifteen sex- specifc genes and many sex-based divergent shared genes (gametologs) are present within this region. Thus far, such genes have not been identifed in natural populations of this species. Results: During a recent feldwork in Taiwan, we encountered natural populations of V. carteri that had not previ‑ ously been recorded from Taiwan. In total, 33 strains of this species were established from water samples collected in Northern Taiwan. Based on sequences of the internal transcribed spacer 2 region of nuclear ribosomal DNA and the presence of asexual spheroids with up to 16 gonidia, the species was clearly identifed as V. carteri f. nagariensis. However, the sexual male spheroids of the Taiwanese strains generally exhibited a 1:1 to > 50:1 ratio of somatic cells to androgonidia. We also investigated the presence or absence of several sex-specifc genes and the sex-based divergent genes MAT3m, MAT3f and LEU1Sm. We did not identify recombination or deletion of such genes between the male and female mating-type locus haplotypes in 32 of the 33 strains. In one putative female strain, the female-specifc gene HMG1f was not amplifed by genomic polymerase chain reaction. When sexually induced, apparently normal female sexual spheroids developed in this strain. Conclusions: Male spheroids are actually variable within V. carteri f. nagariensis. Therefore, the minimum ratio of somatic cells to androgonidia in male spheroids and the maximum number of gonidia in asexual spheroids may be diagnostic for V. carteri f. nagariensis. HMG1f may not be directly related to the formation of female spheroids in this taxon. Keywords: Mating-type locus, Morphology, Sexual reproduction, Taxonomy, Volvox, Volvox carteri, Volvox carteri f. nagariensis Background multicellularity and sex (Kirk 1998; Hiraide et al. 2013). Te genus Volvox represents the most complex mem- In recent years, Volvox carteri f. nagariensis has been ber of volvocine green algae, especially in terms of studied extensively at the cellular and molecular level (e.g., Kirk et al. 1999; Ferris et al. 2010). Although this *Correspondence: [email protected]‑tokyo.ac.jp taxon or form was originally described based on a natu- 1 Department of Biological Sciences, Graduate School of Science, ral sample collected in Nagari, India (Iyengar 1933), most University of Tokyo, Hongo, Bunkyo‑ku, Tokyo 113‑0033, Japan of the studied strains are Japanese strains of V. carteri f. Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Nozaki et al. Bot Stud (2018) 59:10 Page 2 of 8 nagariensis such as Eve (Starr 1969; Kirk et al. 1999; Fer- the mating-type locus genes has not been examined in ris et al. 2010; Geng et al. 2014). Six forms are recognized natural populations of V. carteri. within V. carteri, and the most distinctive morphological During a recent feld survey of the freshwater green attribute for distinguishing V. carteri f. nagariensis from algae in Taiwan, we encountered natural populations of other forms within this species is the production of sex- V. carteri, which have not previously been recorded in ual male spheroids with only a 1:1 ratio of somatic cells Taiwan (Yamagishi 1992). Based on the internal tran- to sperm packets or androgonidia (sperm packet initials) scribed spacer 2 region of nuclear ribosomal DNA (Nozaki 1988). However, this ratio has been examined (nuclear rDNA ITS-2), the populations were clearly only in Japanese strains (Starr 1969; Nozaki 1988; Geng identifed as V. carteri f. nagariensis. Tis study was et al. 2014) irrespective of the presence of Indian strains undertaken to reveal morphological details of V. carteri from Poona (Adams et al. 1990). f. nagariensis originating from Taiwan and to examine Volvox carteri f. nagariensis is an oogamous species presence or absence of several sex-specifc and sex-based that has heterothallic sexuality determined by the sex- divergent genes in these natural populations of V. carteri determining chromosomal region or mating-type locus f. nagariensis. where the presence of sex-specifc genes (genes harbor- ing in only one of the two sexes) and sex-based diver- Methods gent shared genes (gametologs; two homologous genes Table 1 shows the origins of the 33 Taiwanese strains harboring in this regions of both of the two sexes) was used in the present study. Clones were established by the recently demonstrated (Ferris et al. 2010). Sex in V. cart- pipette-washing method (Pringsheim 1946). Te cultures eri f. nagariensis is determined based on the presence or were maintained in screw-cap tubes (18 × 150 mm) con- absence of such genes. However, sex identifcation in nat- taining 11 mL AF-6 or VTAC medium (Kawachi et al. ural populations of this species has not previously been 2013) at 25 °C with a 14:10 h light:dark (LD) schedule conducted by using molecular markers. Furthermore, the under cool-white fuorescent lamps at an intensity of mating-type locus of V. carteri f. nagariensis is composed 80–130 μmol m−2 s−1. Several of these new wild strains of a > 1 Mbp linear chromosome where recombination is are available from the Microbial Culture Collection at the suggested to be repressed (Ferris et al. 2010). Although National Institute for Environmental Studies (Kawachi the mating-type locus or sex chromosomal region does et al. 2013; http://mcc.nies.go.jp/index_en.html) as not exhibit recombination in Chlamydomonas rein- NIES-4205–4210 (Table 1). To observe the morphology hardtii under the laboratory conditions, recombination of asexual spheroids, the cultures were grown in 11 mL and gene conversion of C. reinhardtii mating-type locus VTAC medium in Petri dishes (10 × 60 mm) at 25 °C genes were demonstrated in natural populations of this with a 14:10 h LD cycle. For comparison, we also used species (De Hof et al. 2013). However, recombination of Eve [i.e., UTEX 1885, obtained from the Culture Collec- tion of Algae at the University of Texas at Austin (UTEX, Table 1 List of Taiwanese strains of Volvox carteri f. nagariensis Habitat/date [locality Longitude, latitude Number of Sex based on Strains deposited in NIES-col- designation] [temperature/pH] strains genomic PCR lection and their DDBJ/EMBL/ GenBank accession numbers of nuclear rDNA ITS-2 Rice paddy/25 May 2016 [2] N 25°13′02.76′′, E 0 Male 121°36′22.44′′ [26.1 °C, pH 1 Female 2016‑tw‑nuk‑Chg ( NIES‑4205), 6.7] LC376031 = Fallow rice paddy/9 Jun 2016 N 25°13′51.27′′, E 4 Male [4] 121°37′53.78′′ [36.2 °C, pH 12 Female 2016‑0609‑v‑1 ( NIES‑4206), 6.8] LC376032 = Fallow rice paddy/10 Jun N 25°00′52.51′′, E 14 Male 2016‑0610‑v‑11 ( NIES‑4207), 2016 [6] 121°55′33.38′′ [29.7 °C, pH LC376033; 2016=‑tw‑nuk‑6‑1 6.7] ( NIES‑4208), LC376034 = 0 Female Rice paddy/10 Jun 2016 [8] N 25°01′09.82′′, E 1 Male 2016‑tw‑nuk‑8‑1 ( NIES‑4209), 121°55′21.76′′ [29.0 °C, pH LC376035 = 5.7] 1 Female 2016‑tw‑nuk‑8‑2 ( NIES‑4210), LC376036 = Nozaki et al. Bot Stud (2018) 59:10 Page 3 of 8 https://utex.org)], a typical female strain of V. carteri f. is sister to V. carteri (Kawafune et al. 2015; Nozaki et al. nagariensis (Ferris et al. 2010; Geng et al. 2014). 2015). Morphological details of asexual and sexual spheroids We performed genomic polymerase chain reaction were observed mainly in three strains, 2016-0609-v-1 (PCR) using the primers listed in Table 2 to check the (female), 2016-tw-nuk-6-1 (male) and 2016-tw-nuk-8-1 presence or absence of several sex-specifc genes and the (male). To observe asexual spheroids and reproduc- sex-based divergent genes MAT3m, MAT3f and LEU1Sm tion, we examined small aliquots of asexual spheroids, in (Ferris et al. 2010) in the 33 Taiwanese strains and Eve. actively grown 2- to 5-day-old cultures in the Petri dishes PCR was carried out using KOD FX Neo DNA polymer- (10 × 60 mm). Sexual male spheroids developed spon- ase (TOYOBO, Osaka, Japan) according to the manufac- taneously in a culture of male strains grown in VTAC turer’s instructions. medium at 20 °C. To induce sexual spheroids, 0.1–0.2 mL of the sexual inducer (supernatant of the male culture Results after the formation of sperm packets) and ca.