Materials and methods

Taxonomic treatment. Specimens were legally collected according to the scientific permit SPARN/DGVS/08149/23. We examined the putative new species and compared with all morphologically similar Pinguicula specimens from the ENCB, FCME, IBUG, IEB, MEXU, OAX, SERO, UAMIZ, and XAL herbaria (acronyms according to ^{Thiers 2024}). The morphological description was elaborated based on herbarium specimens and living plants. The terminology followed ^{Casper (1966)}, ^{Moreno (1984)}, ^{Zamudio (2001)}, and ^{López-Pérez (2017)}. The colour of the structures was designated in accordance with the RHS Colour Chart (^{Royal Horticultural Society 2015}).

Conservation status. The conservation status of the new species was evaluated based on the IUCN Red List Criteria and Categories (^{IUCN 2022}). The Extent of Occurrence (EOO) and Area of Occupancy (AOO) were estimated with the GeoCAT tool (^{Bachman et al. 2011}).

Plant material, DNA extraction, amplification, and sequencing. The sampling comprised eight Pinguicula species from the clade VII of the nuclear phylogenetic hypothesis of ^{Shimai et al. (2021)}. This clade includes morphologically similar Pinguicula species to the putative new species. Taxa were selected to represent the subclades within the clade. The study included 16 accessions available in GenBank. Further, fresh material of P. acuminata Benth., P. emarginata Zamudio & Rzed., P. heterophylla Benth., P. medusina Zamudio & Studnička, P. parvifolia B.L.Rob., and the putative new taxon were collected. The samples were processed to generate herbarium specimens and leaves were dried in silica gel for DNA extraction according to ^{Funk et al. (2017)}. Genlisea lobata Fromm, G. violacea A.St.-Hil., Utricularia foliosa L., U. floridana Nash, and U. gibba L. were employed as outgroups. The accession and collection numbers of these are listed in Appendix 1. The specimens were deposited in the IBUG herbarium.

DNA extraction was carried out with the protocol of ^{Doyle & Doyle (1987)}. The chloroplast intergenic spacers rpL32-trnL, trnQ-5´rps16 (^{Wicke et al. 2014}, ^{Shaw et al. 2014}), and the Internal Transcribed Spacer (ITS, ^{Cheng et al. 2016}) were amplified and sequenced (Table 1). PCRs were performed in a reaction mix of 8.5 µL, including 0.5 µL DNA (~ 50 ng/ µL), 0.5 µL of each primer [10 mM], 3.0 µL of DreamTaqTM Green PCR Master Mix (2x), and 4.0 µL of RNase-Free Water. The amplification parameters for the rpL32-trnL and trnQ-rps16 included: 1) 80 °C for 5 min for an initial denaturation, 2) 30 cycles of denaturation at 95 °C for 1 min, first annealing at 50 °C, followed by a 0.3 °C ramp to 65 °C for 1 min and extension at 65 °C for 4 min, 3) a final extension at 65 °C for 5 min. For ITS, the PCR parameters were: 1) 80 °C for 5 min for an initial denaturation, 2) 30 cycles of denaturation at 95 °C for 1 min, an alignment at 52 °C for 1 min, an extension of 65 °C for 4 min, 3) a final extension at 65 °C for 4 min. DNA extraction, amplification, and sequencing were carried out in the Laboratorio Nacional de Identificación y Caracterización Vegetal (LaniVeg) of the Universidad de Guadalajara.

Phylogenetic analyses. Phylogenetic relationships were estimated with three data sets: rpL32-trnL + trnQ-rps16, ITS, and rpL32-trnL + trnQ-rps16 + ITS. We used Bayesian Inference (BI) and Maximum Likelihood (ML) methods. The Utricularia-Genlisea clade was defined as the outgroup (^{Jobson & Albert 2002}, ^{Müller et al. 2004}, ^{Shimai et al. 2021}). The best molecular evolution models were estimated for each partition with jModelTest v. 2.1.10 (^{Posada 2008}). Thus, the best evolutionary model was inferred for each intergenic spacer, and the best-of-fit DNA models were selected based on the corrected Akaike Information Criterion (^{AICc, Akaike 1998}, ^{Burnham & Anderson 2002}). BI analyses was performed using MrBayes v. 3.2.2 (^{Ronquist et al. 2012}) with 50 × 10⁶ generations and a sampling frequency of 1,000. The first 25 % of the trees were discarded as burn-in to ensure that the chains reached stationarity. Posterior probabilities (PP) were obtained by a majority-rule consensus tree (> 50 %). For the ML analyses, we employed RAxML HPC BlackBox (^{Stamatakis 2014}) tool in CIPRES Science Gateway v. 3 portal (^{Miller et al. 2010}). We used the option Let RAxML halt bootstrapping automatically. The likelihood of the final tree was estimated under the GTR + G + I model (^{Stamatakis 2006}, ^{Yang 1993}). Cladograms were drawn with FigTree v. 1.4.4 347 software (^{Rambaut 2018}).

Database and species richness distribution of Pinguicula gypsophytes in Mexico. We included all the gypsophytes species of Pinguicula in Mexico considering the definition of ^{Pérez-García et al. (2017)}, who mention that a gypsophyte is a plant species growing exclusively (or almost) on gypsum soils. The taxa were selected based on the information attached to herbarium specimens, digital herbaria, digital databases and original descriptions. The Pinguicula records from CAS (digital), CHAP, CHAPA, CIIDIR, ENCB, IBUG, IEB, INEGI, MEXU, MO (digital), SLPM, UAMIZ, XAL, and ZEA herbaria (acronyms according to ^{Thiers 2024}), the information available in the Global Biodiversity Information Facility (^{GBIF 2023}), and the Southwest Environmental Information Network (^{Gilbert et al. 2019}) were compiled and curated to construct the database and species distribution maps. To assure the correct taxonomic identity of the digital records, only specimens with images were examined. Specimens without geographical data were georeferenced using Google Earth Pro v. 9.191.0.0 (^{Google 2023}) and the Mapa Digital de México v. 6.1 (^{INEGI 2023}) following the recommendations of ^{Garcia-Milagros & Funk (2010)}. Specimens with ambiguous information of location were excluded.

The species richness distribution of Pinguicula gypsophytes in Mexico was estimated by 1) state, 2) physiographic province, and 3) 1 × 1° cells. The species richness distribution analysis by state was carried out through a direct count of species documented in each polygon according to ^{INEGI (2018)} limits. We used the limits established by ^{Ferrusquía-Villafranca (1993)} for the species richness analysis by physiographic provinces, while for the grid cell richness analysis we employed a cell size of 1 × 1°.

Results

Pinguicula tonalaensis López-Pérez & Zamudio sp. nov. (Figures 1 and 2, Table 2).

Illustrated by Ericka Belén Cortez Castro based on type (J. López-Pérez & G. Munguía-Lino 680) and fresh material.

Figure 1 Pinguicula tonalaensis. A) Winter rosette; B) summer rosette; C) winter leaves; D) summer leaves; E) surface of summer leaves; F) trichomes of leaves, peduncle, calyx and corolla tube surface; G) calyx; H) frontal corolla view; I) trichomes of the surface corolla lobes; J) lateral view of corolla tube variation; K) fruit mature; L) seeds.  

Photographs of Jorge López-Pérez.

Figure 2 Pinguicula tonalaensis. A) Winter rosette; B) summer rosette; C) winter leaves; D) base of the summer leaves; E) surface of summer leaves; F) variation in the corolla tube; G) dorsal and ventral view of the corolla tube; H) ventral and dorsal view of the calyx, and mature fruit; I) seeds. A-I)  

Type. Mexico, Oaxaca, municipio Santo Domingo Tonalá, ± 3.5 km al suroeste de Tonalá (Santo Domingo), 1,416 m asl, 01 August 2023, J. López-Pérez & G. Munguía-Lino 680 (Holotype: IBUG; Isotypes: MEXU, OAX).

Diagnosis. Pinguicula tonalaensis shares winter and summer leaf forms and size with P. medusina. The geniculate corolla tube and the lack of apical propagules on the summer leaves set P. tonalaensis apart.

Description. Perennial herbs. Leaves dimorphic, arranged into basal rosettes; winter rosette compact, hypogeous, 7.5-12.0 mm in diameter, leaves 55-70, succulents, sessile, 8.0-18.0 × 1.2-3.0 mm, elliptic-lanceolate, acuminate, glabrous; summer rosette lax, leaves 7-12, sessile, erect, 3.5-15.5 cm × 1.6-4.5 mm, linear, acuminate, margin revolute, pubescent on the upper surface with sessile and stipitate glandular trichomes, ciliate at the base, cilium ca. 4.5 mm, without apical propagules. Scapes 3-5 per plant, 5.5-12.0 cm long, pilosulous with glandular trichomes. Flowers (14.0)16.5-22.5 mm long including the spur. Calyx bilabiate, outer surface pilosulous with stalked glandular trichomes; upper lip trilobate, triangular lobes, 1.5-2.5 × 0.5-1.0 mm; lower lip bilobate, lobes triangular to lanceolate, 2.0 × 0.5 mm. Corolla subisolobate, white, with a Strong Yellow-Green (Yellow-Green Group N144-C) macula at the base of the lips that extends to the throat, lobes obovate to obovate-oblong, apex rounded to truncate, pilosulose; upper lip bilobate (3.0)4.5-6.5 × 2.5-4.0(5.5) mm; lower lip trilobate, lateral lobes 5.0-7.7 × 2.0-4.2 mm, the medium larger than the laterals ones, 5.3-9.0 × 2.5-5.0 mm. Corolla tube geniculate, Strong Yellow-Green to Deep Purple (Yellow-Green Group N144 B, Violet Group 83 A), 5.0-8.5 × 2.5-4.0 mm, pilosulose with stalked glandular trichomes. Spur tubular, 2.6-6.0 mm long, Strong Yellow-Green to Brownish Orange (Yellow-Green Group N144 C, Greyed-Orange Group 165 B), acute, occasionally emarginated. Capsule globose, ± 2.0 mm in diameter, pilosulose. Seeds elliptic, 0.5 × 0.2 mm, reticulate, apiculate.

Distribution and ecology. Pinguicula tonalaensis grows in the municipality of Santo Domingo Tonalá, Oaxaca (Figure 3). The area is part of the Sierra Madre del Sur physiographic province (^{Ferrusquía-Villafranca 1993}). It inhabits northeastern facing gypsum ravines and hills at 1,416 m asl, covered by xeric scrubland. The plants grow in sympatry with Begonia tlapensis Burt-Utley & Utley, Calochortus multicolor García-Mend., D.Sandoval & C.Chávez, Fouquieria ochoterenae Miranda, Physodium corymbosum C.Presl, Polystemma calcicola (Greenm.) Morillo, Dahlia sp., and Sellaginella sp.

Figure 3 Distribution of Pinguicula gypsophytes in Mexico. A) Species richness distribution; B) species in the Sierra Madre Oriental; C) species in the Oaxaca State. Solid black line: physiographic limit. Grey line: state limit. Solid dot line: municipal limit in Oaxaca state. 

Conservation status. Pinguicula tonalaensis is known from three localities in municipality of Santo Domingo Tonalá, Oaxaca. GeoCAT calculated an EOO of 0.045 km² and an AOO of 8.0 km² (based on a cell width of 2 km). According to the IUCN Red List guidelines and criteria B1 and B2, we preliminary recommend the category of Critically Endangered (CR), criterion B1ab(iii)+2ab(iii).

Phenology. The plants bloom from July to September. Flowering occurs when the summer leaves are completely developed. The summer leaves survive until November, then they shrivel to the winter rosette. The winter rosette remains underground during the dry season and the summer rosette emerges at the beginning of the rainy season of the following year.

Etymology. The specific epithet refers to Santo Domingo Tonalá municipality, where the plants grow.

Additional specimens examined. Mexico, Oaxaca, municipio Santo Domingo Tonalá, carretera 15 (tramo Río Santiago Copala a Juxtlahuaca), ± 2 km al suroeste de Yetla de Juárez, 1,449 m asl, 23 September 2024, J. López-Pérez & G. Munguía-Lino 767 (IBUG, IEB, MEXU); Carretera 15 (tramo Santo Domingo Tonalá a San Agustín Atenango), 1,437 m asl, 23 September 2024, J. López-Pérez & G. Munguía-Lino 768 (IBUG, IEB, MEXU).

Phylogenetic relationships. Three genera, 15 species, and 37 sequences were employed in the phylogenetic analyses (Appendix 1). This included 26 Pinguicula samples. The data set had a total length of 1,637 bp for rpL32-trnL + trnQ-rps16, 901 bp for ITS, and 2,537 bp for rpL32-trnL + trnQ-rps16 + ITS. The corrected AIC criterion estimated the model TPM1uf + G for rpL32-trnL, TPM3uf for rps16-trnQ, and GTR + I + G for ITS. The results of the analyses of the three data sets showed P. tonalaensis as the sister group of the clade formed by P. heterophylla and P. medusina (PP ≥ 0.84, BS ≥ 79 %; Figure 4).

Figure 4 Phylogenetic relationships of Pinguicula tonalaensis. The numbers in the nodes show the posterior probabilities (PP) and the bootstrap support (BS %). Nodes with support ≥ 70 % are indicated. 

Pinguicula gypsophytes in Mexico. There are 10 gypsophyte Pinguicula species in Mexico (Figure 5, Table 3), and all of them are endemic to the country. These species can be identified as follows:

1. Corolla isolobate to subisolobate, lobes equal or almost equal between the superior and inferior lips ............ 2

2. Annual plants; palate minute, pubescent ................................................................................... P. takakii

2. Perennial plants; palate absent ........................................................................................................... 3

3. Winter rosette hypogeal, compact; summer leaves linear-lanceolate ........................................................ 4

4. Corolla tube straight; with propagules in the apex of the summer leaves ................................... P. medusina

4. Corolla tube geniculate; without propagules in the apex of the summer leaves ......................... P. tonalaensis

3. Winter rosette epigeal, lax; summer leaves cuneate or spathulate ............................................................ 5

5. Corolla lobes with evident purple veins; base of the lower corolla lip covered with yellow trichomes ............................................................................................................................................... P. kondoi

5. Corolla lobes with inconspicuous veins or the same colour as the lobes; base of both corolla lips covered with white trichomes .................................................................................................................. P. rotundiflora

1. Corolla lobes with a clear distinction between the superior and inferior lips ................................................ 6

6. Annual plants ..................................................................................................................... P. pygmaea

6. Perennial plants ................................................................................................................................ 7

7. Corolla white; spur shorter than the corolla ........................................................................................... 8

8. Upper corolla lobes obovate, ondulate, 3-7 × 3-6 mm .................................................................. P. nivalis

8. Upper corolla lobes oblong, entire, 1-3 × 1.0-1.5 mm .......................................................... P. immaculata

7. Corolla pink or purple; spur larger than the corolla ................................................................................. 9

9. Summer leaves linear-lanceolate; corolla lobes oblong ……........................................................ P. gypsicola

9. Summer leaves elliptic to suborbicular; corolla lobes suborbicular to oblate ............................... P. colimensis

Photographs by Jorge López-Pérez.

Figure 5 Ggypsophyte species of Pinguicula in Mexico. A) P. colimensis; B, F) P. gypsicola; C, G) P. kondoi; D, H) P. medusina; E) P. pygmaea; I, M) P. nivalis; J, N) P. rotundiflora; K, O) P. takakii; L, P) P. tonalaensis. A-P)  

Species richness distribution of Pinguicula gypsophytes in Mexico. The analyses showed that gypsophyte Pinguicula species are present in the states of Colima, Nuevo León, Oaxaca, San Luis Potosí, and Tamaulipas (Figure 3, Table 3). Nuevo León had the most species, where P. immaculata, P. kondoi, P. nivalis, and P. rotundiflora were registered (Figure 3B). Colima and Tamaulipas had one species each, P. colimensis and P. rotundiflora. The gypsophyte species are restricted to the physiographic provinces of Sierra Madre Oriental (SMOr) and Sierra Madre del Sur (SMS) (Figure 3). The SMOr included six species: P. gypsicola, P. immaculata, P. kondoi, P. nivalis, P. rotundiflora, and P. takakii (Figure 3A, B, Table 3). The SMS had four species: P. colimensis, P. medusina, P. pygmaea, and P. tonalaensis (Figure 3A, C). The grid cell analysis placed the cell with the highest species values on the SMOr (Figure 3A, B).

Discussion

Taxonomic treatment. According to the classification of ^{Casper (1966)}, the new taxon pertains to Pinguicula subg. Isoloba. The presence of winter and summer rosettes, the isolobate corollas, the subcylindric corolla tube, and the spur shorter than the corolla tube characterize the subgenus. Pinguicula tonalaensis is morphologically similar to P. heterophylla and P. medusina (Figure 6, Table 2). The morphology of the winter rosette in P. heterophylla, P. medusina, and P. tonalaensis is similar. It is composed of thick underground leaves strongly grouped together. These are covered by the dry ciliate base of the summer leaves from the previous growing season. This structure resembles a tunicate bulb (Figures 1A, 2A). However, P. tonalaensis differs from P. heterophylla by the absence of propagules in the apex of summer leaves and by growing up on gypsum soils covered with xeric scrubland. In contrast, P. heterophylla develops propagules in the apex of summer leaves and grows in pine-oak forest. The new species is more similar to P. medusina, but differs by the absence of propagules at the apex of summer leaves. In addition, the presence of a geniculate corolla tube in P. tonalaensis distinguished it from both species (Figure 6C, G, K). ^{Zamudio & Studnicka (2000)} considered that P. medusina develops propagules in the apex of summer leaves, whereas P. heterophylla lacks them. The authors highlight the absence of propagules in P. heterophylla as a taxonomic character. However, observations in the field and cultivated plants show that this taxon does develop apical foliar propagules in the summer leaves too (Figure 6D).

Photographs by Jorge López-Pérez; C) photograph by Erick Vélez-Sánchez.

Figure 6 Morphologically comparison of Pinguicula tonalaensis and similar species. A-D) P. heterophylla; E-H) P. medusina; I-L) P. tonalaensis; A, E, I) habitat; B, F, J) frontal corolla view; C, G, K) lateral corolla view; D, H) propagules in P. heterophylla and P. medusina respectively; L) summer leaves without propagules in P. tonalaensis. A-B, D-L)  

Species richness distribution of gypsophyte Pinguicula in Mexico. The Mexican Transition Zone (MTZ) represents the boundary between the Nearctic and Neotropical regions (^{Villaseñor et al. 2020}). It is a set of morphotectonic and physiographic provinces with different ages and origins (^{Ferrusquía-Villafranca 1993}, ^{Mastretta-Yanes et al. 2015}). Within the MTZ, the SMOr represents an area of high species richness and endemism for Pinguicula (^{Zamudio 2005}, ^{Salinas-Rodríguez et al. 2017}, ²⁰²², ^{Domínguez et al. 2024}, ^{López-Pérez et al. 2024b}). A similar pattern for other groups was observed by ^{Contreras-Medina & Luna-Vega (2007)}, ^{Torres-Miranda et al. (2011)}, ^{Luna-Vega et al. (2013)}, ^{Sanginés-Franco et al. (2015)}, and ^{Tellez et al. (2020)}, who emphasized that the taxonomic richness of gymnosperms, oaks, vascular plants, and native trees of Mexico and Central America is centred in this province. Concerning species richness and endemism of gypsophytes in Mexico, ^{Ortiz-Brunel et al. (2023)} also highlighted this province as the richest. Based on grid cell analyses, they identified the Cuatro Ciénegas Basin in Coahuila as the richest area and as an Endemism Centre. Also they highlight the relevance of the surroundings of Santo Domingo Tonalá in Oaxaca, for its elevated number of restricted species. Finally, the description of this new gypsophyte species from Oaxaca increases to 55 the number of Pinguicula species in Mexico. This work also emphasizes the importance of gypsum soils for the formation of endemic species of Pinguicula.