Allium pseudotelmatum M. Duchoslav & M. Jandová, 2017

Jandová, Michaela, Oulehlová, Martina, Vojtěchová, Kateřina, Kitner, Miloslav & Duchoslav, Martin, 2017, Allium pseudotelmatum (Amaryllidaceae), a new species of Allium sect. Codonoprasum from Croatia, Phytotaxa 311 (2), pp. 139-154 : 141-148

Allium pseudotelmatum M. Duchoslav & M. Jandová , sp. nov. ( Fig. 1 , 2 )

Diagnosis:—Similar to A. telmatum , but differing in having bulbils within the inflorescence, larger flowers with white tepals tinged with pinkish to pink-purplish, longer stamens and bigger anthers and ovary, white style, 17–18 leaf vascular bundles, two to three layered leaf palisade tissue, earlier flowering period, and different habitat.

Type:— CROATIA. South Dalmatia : Opuzen, cultivated Citrus reticulata plantations near the town, elevation 5 m, coordinates WGS-84: 43°00´8.6˝N 17°33´03.0˝E, 13 August 2016, Duchoslav M. & Jandová M. (holotype, OL! [cultivated plants, herbarium specimen, accession number 33960]; isotype, BRNM!) .

Description: —Bulb ovoid, 15−20 × 12−17 mm, inner tunics whitish, membranous, outer tunics coriaceous, browngreyish ( Fig. 1H ). Bulblets 1–2, ovoid, 5–10 × 5–15 mm, white or yellowish, with contractile roots and 1–3 leaves, on 1–7 cm long axils growing belowground ( Fig. 1H ). Stem 30−45 cm tall, cylindrical, glabrous, erect, covered by leaf sheaths along the basal third to half of its length ( Fig. 1A ). Leaves 3−5, green, 5–ribbed (elongated pentagonal in the cross section; Fig. 3A ), 8.0− 19.5 cm long, 2.0− 2.8 mm wide, with papillose edge ( Fig. 3C ) and denticulate epidermis ( Fig. 4A ). Spathe reflexed to suberect, persistent, with unequal valves, the larger one longer than the inflorescence, the larger 4−14 cm long, the smaller 3.5−7.5 cm long ( Fig. 1A, B ). Inflorescence lax and fastigiate, containing 30−52 flowers and 10–52 bulbils at the base of pedicels ( Fig. 1A, B ). Pedicels unequal, 10−40 mm long. Perigon cup-shaped to campanulate, with monomorphic tepals, white, tinged with pinkish to pink-purplish, obovate-elliptical, apiculate at the apex, 6.0−7.0 mm long, the inner ones 3.3–3.8 mm wide, the outer ones 3.1−4.0 mm wide, with dirty purplish midrib ( Fig. 1C , 2A, B ). Stamens exerted from the perigon with simple filaments, purplish above and white below ( Fig. 1B, C, F , 2A ), unequal, the outer ones 3.4−6.6 mm long, the inner ones 4.5−6.6 mm long, all connate below into an annulus 1.2−1.5 mm high; anthers white-yellowish, oblong-elliptical, 1.6−1.8 × 0.9−1.0 mm, rounded at the apex ( Fig. 1F ). Ovary obovoid, narrowed at the base, green, tuberculate above, 4.4−4.9 × 2.1–2.6 mm; style white, 1.1−4.0 mm long ( Fig. 1D ). Capsule trivalved, obovoid, 5.2−6.2 × 4.4−4.6 mm, green ( Fig. 1E ). Seeds elongated, curved like a banana, 4.5–5.2 × 1.7–2.3 mm, with black testa. Bulbils ovate, stipitate (1–3 mm long) and with long narrow (caudate) apex (1–3 mm long), dirty white when young, green-purplish in maturity ( Fig. 1G , 2D ).

Leaf anatomy: —The leaf cross section of A. pseudotelmatum shows elongated pentagonal outline ( Fig. 3A ). The one-layered epidermis is covered by a well-developed cuticle. On the surface of leaf epidermis, each cell has micropapillae arranged into rows and longitudinal laths connecting the papillae in adjacent cells are missing or are only suggested. Transversal cell walls at the point of contact of adjacent epidermal cells are obvious between the cell walls and transversal grooves ( Fig. 4A ). The sunken stomata are numerous and they are distributed along the whole perimeter ( Fig. 3A, C ). The palisade tissue is regular and compact, and arranged in two-layered prosenchymatical cells; at some points it is three-layered; the outer layer usually has bigger and elongated prosenchymatical cells ( Fig. 3D ). The spongy tissue is quite compact with more or less irregular big cells. In the peripheral part of the spongy tissue several secretory canals occur. There are 17–18 colateral closed vascular bundles with sclerenchymatical bundle-sheat around xylem ( Fig. 3E ), of which 10–11 are abaxial (three are larger) and 7 are adaxial (four are larger) ( Fig. 3A ).

Variability: —Number of bulbils varies from ca. 10 to 50 per plant, depending positively on the size of a plant.

Karyology: — Allium pseudotelmatum is a pentaploid plant with a chromosome complement 2n = 5x = 40 ( Fig. 5A ). Holoploid genome size (2C; mean ± SD, n = 6) is 63.7 ± 0.2 pg and monoploid genome size (1Cx) is 12.8 pg. The karyotype is regular and homogeneous, characterized by 34 of more-or-less metacentric chromosomes (including four microsatellites) and 6 submetacentric chromosomes. The microsatellites were observed on the short arms of two chromosomes and on the long arms of two other chromosomes. Thus, the chromosome formula is 2n = 40: 2 M + 28 m + 4 msat + 6 sm ( Fig. 5B ). Absolute chromosome length varies from 4.7 ± 0.1 μm for the shortest chromosome to 9.1 ± 0.1 μm for the longest one. The arm ratio varies from 1.02 to 1.84.

Phenology: —The new species flowers in late summer, ranging from early August to September. Seed ripening period ranges from middle September to early October when most seeds usually are released from the capsule. Aerial bulbils are clustered within the inflorescence and gradually fall out during October. During flowering period, when leaves are almost withered, plants produce 1–2 new leaves from the bulb that survive winter; therefore plants are not dormant. This ontogenetic cycle was also observed on cultivated plants during two years of cultivation in the common garden.

Habitat and ecology: — Allium pseudotelmatum grows in cultivated Citrus reticulata plantations. It forms dense clusters beneath canopy of trees due to underground vegetative reproduction through production of 1–2 underground daughter bulbs (bulblets) on short stolons (1–7 cm long). Soil is regularly weeded and sprayed against weeds. Thanks to artificial irrigation, soils are freshly moist and probably well supplied with nutrients.

Distribution: —So far, A. pseudotelmatum has been recorded in the floodplain of the Mala Neretva River, southwestern from the town of Opuzen in South Dalmatia ( Croatia), where it occurs sparsely in cultivated Citrus reticulata plantations. We suppose that more intensive search could discover more localities in the surroundings in similar habitats.

Conservation status: —Ruderal character of A. pseudotelmatum sites connected with relative frequent occurrence of the species even under heavy cultivation suggests that the species is probably able to withstand disturbances caused by agricultural practices. In one site the population includes hundreds of plants. So far, the species range sums up to less than 1 sq. km. However, to precisely evaluate its real conservation status, appropriate data on distribution are needed. Thus, based on the IUCN criteria (2012), it is proposed to include it in the following category: data deficient (DD).

Etymology: —The specific epithet is given considering its similarity with A. telmatum .

Taxonomic affinities: —The occurrence of two-valved persistent spathe with unequal valves, longer than the pedicels, simple stamens and an ovary with inconspicuous nectaries ( Stearn 1980) place A. pseudotelmatum into the sect. Codonoprasum . Within this section, Allium pseudotelmatum appears morphologically and phenologically similar to the group of the autumn-flowering species ( Brullo et al. 1994, 1997b, 1997c, Bogdanović et al. 2009) but due to overall habit and the presence of bulbils within the inflorescence it can be also confused with the summer-flowering A. oleraceum . However, a series of character-state combinations allow a clear differentiation among them. Below we discuss differentiation of A. pseudotelmatum from the abovementioned species from various points of view.

A common feature of the autumn-flowering species of the sect. Conodoprasum is the almost absence of dormancy period when new leaves are produced by newly renewed bulb and sprout aboveground during the flowering time in late summer and autumn (September–October). Such peculiar ontogenetic cycle with almost continual vegetative phase differs from the early summer-flowering representatives of the section (e.g., A. oleraceum , A. paniculatum Linnaeus (1759: 978) , and A. fuscum Waldstein & Kitaibel (1808: 267)) whose newly renewed bulb sprouts during late autumn after one-two months of the resting period ( Duchoslav 2009, Duchoslav & Jandová, pers. obs.). While A. pseudotelmatum has also almost continual vegetative phase, it differs from the other members of the autumn-flowering species group but also from A. oleraceum by an “intermediate” flowering period that starts during August when A. oleraceum finishes flowering ( Duchoslav 2009), and finishes in September when species of the autumn-flowering group usually start flowering. Allium pseudotelmatum plants retained flowering time period during two years of cultivation in a common garden. Similar “intermediate” phenology has been recently observed in A. orestis Kalpoutzakis et al. (2012: 196) , endemic of Peloponnisos.

Another difference regards to its habitat. In fact, members of the autumn-flowering group inhabit following natural habitat types ( Kollmann et al. 1991, Tzanoudakis & Kypriotakis 1993, Brullo et al. 1994, 1997a, 1997b, 1997c, Tzanoudakis 2000, Biel et al. 2006, Bogdanović et al. 2009, Kalpoutzakis et al. 2012, Rey et al. 2015): (i) coastal damp marches ( A. savii , A. telmatum ), (ii) undergrowth of deciduous or evergreen forests (e.g., A. euboicum Rechinger (1961: 435) , A. rausii Brullo et al. (2003b: 136) , A. tardiflorum and A. anzalonei ) and (iii) open or shady rocks or dry grassy slopes (e.g., A. tardans Greuter & Zahar. in Zahariadi (1975: 51), A. platakisii , A. archeotrichon Brullo et al. (1999: 42) , A. aegilicum and A. oporinanthum ). Allium pseudotelmatum inhabits arable land, i.e. underwood of cultivated orchards with soils well supplied with nutrients and moisture and its niche is thus rather distinct from most autumn-flowering species but overlap with that of A. oleraceum (wide ecological amplitude including cultivated land; Stearn 1980, Duchoslav 2001, Duchoslav et al. 2010, Haeggström & Åström 2005) and slightly also with that of A. savii and A. telmatum (open stands with fresh moist soils; Bogdanović et al. 2009).

Other differences between A. pseudotelmatum and representatives of the autumn-flowering group regard chromosome number. While A. pseudotelmatum is pentaploid (2n = 40), all known western Mediterranean species including A. telmatum and A. savii are tetraploid (2n = 32) and all but one ( A. apolloniensis Biel et al. (2006: 367) ; 2n = 32) eastern Mediterranean species are diploid (2n = 16), respectively ( Brullo et al. 1994, 1997b, 1997 c, 2014, Biel et al. 2006, Bogdanović et al. 2009). Such unusual ploidy level is known to occur in several summer-flowering polyploid species of the section Codonoprasum , including A. oleraceum and A. dentiferum , species of probably allopolyploid origins with known six ( Šafářová & Duchoslav 2010, Duchoslav et al. 2013) and two ( Brullo et al. 2008) ploidy levels, respectively.

Based on morphological similarities in overall habitus, inflorescence type and flower shape, A. pseudotelmatum shows closer relationships with two autumn-flowering species, i.e. A. telmatum distributed in North Dalmatia ( Bogdanović et al. 2009) and A. savii occurring in Italy, Corse, Sardinia, southern France and Menorca ( Brullo et al. 1994, Fraga 2002, Bogdanović et al. 2009, Tison et al. 2014) but also with common A. oleraceum widely distributed in Europe ( Stearn 1980, Hultén & Fries 1986, Duchoslav 2001) including the northern part of Croatia ( Milović 2015, Nikolić 2016), Serbia and Montenegro ( Tatić 1975, Hadžiblahović 2005). However, A. pseudotelmatum differ from the above-mentioned species in the flowering period, and several karyological, anatomical and morphological characters.

As for the karyotype, both A. pseudotelmatum and A. savii have prevalently metacentric chromosomes, four ( A. pseudotelmatum ) or six ( A. savii ) chromosomes have microsatellites, and six chromosomes are submetacentric ( Fig. 5 , Brullo et al. 1994), while A. telmatum shows only two submetacentric chromosomes, and no evident microsatellites were observed ( Bogdanović et al. 2009). Pentaploids of A. oleraceum have prevalently metacentric chromosomes, from one to three chromosomes have microsatellites and three chromosomes are submetacentric ( Fialová 1996). In addition, chromosome size in A. pseudotelmatum is smaller than in A. telmatum ( Bogdanović et al. 2009) and A. oleraceum ( Fialová 1996) .

Regarding anatomy and morphology of leaf ( Table 1, Fig. 3 , 4 ), leaf cross section of A. pseudotelmatum shows rather flat and elongated pentagon while the shape of leaf cross section of A. telmatum has pentagonal outline ( Bogdanović et al. 2009). Allium savii has flat leaves or semicylindrical narrow leaves with more leaf ribs ( Brullo et al. 1994). On the other hand, leaf cross section of A. oleraceum is rather distinct from abovementioned species, having usually wider leaves with semicylindrical leaf cross section with 5 or more leaf ribs on the abaxial face ( Fig. 3B ; see also Stearn 1980).

The leaf cross section of all compared Allium species is characterized by an epidermis with well-developed cuticle covers and sunken stomata distributed along whole perimeter ( Fig. 3 ; Brullo et al. 1994, Bogdanović et al. 2009). There are significant differences between count of palisade tissue layers and vascular bundles among species. Allium savii has leaves characterized by one-layered palisade tissue with 10–18 vascular bundles (4–6 adaxial and 5–12 abaxial), in A. telmatum leaves there are two-layered palisade tissue and 16−19 vascular bundles (10−11 abaxial and 7−8 adaxial), A. pseudotelmatum has leaves characterized by two-layered but at some points three-layered palisade tissue and 17–18 vascular bundles (10–11 abaxial and 7 adaxial), while A. oleraceum has two-layered palisade tissue and 19–22 vascular bundles (11–13 abaxial and 8–9 adaxial; Fig. 3A, B ). Bogdanović et al. (2009) and Brullo et al. (1994) described in A. telmatum and A. savii leaves with fistulous spongy tissue in the inner part of central portion, while leaves of A. oleraceum are fistulous in the lower part of leaf ( Fig. 3B ) and canaliculate above ( Stearn 1980). In contrast, A. pseudotelmatum leaves has quite compact spongy tissue with more or less irregular big cells.

Seeking for new and easily obtained characters useful for discrimination among species we studied epidermal characters that have been successfully used for identifying closely related species in the genus Allium ( Yousaf et al. 2008, Lin & Tan 2015) including section Codonoprasum ( Zahariadi 1975, Krahulec 1980). In accordance with observations made by Krahulec (1980), we observed epidermal cells of A. oleraceum with micropapillae arranged into rows or dispersed. Micropapillae are frequently connected in the longitudinal direction of leaf into thin laths ( Fig. 4B ) and transversal cell wall at the point of contact of adjacent epidermal cells are indistinct, with inconspicuous transversal grooves in the point of contact of cells. In contrast, A. pseudotelmatum has distinct epidermal characters with micropapillae arranged into rows but with apparent transversal grooves between the cell walls ( Fig. 4A ), which clearly differ from epidermal character of A. oleraceum .

Morphological comparison of A. pseudotelmatum and three similar species is summarised in Table 1. Besides having bigger size of some flower and fruit parts (tepal width, stamens, ovary, and capsule), different colour of tepals, and perianth and spathe shape (see Table 1), A. pseudotelmatum also differs from both A. telmatum and A. savii by the presence of bulbils within the inflorescence. Bulbil production has been related to ploidy level in representatives of Allium with plants bearing bulbils in the inflorescence being polyploid ( Levan 1937, Pastor 1982). However, this ploidy-related pattern of bulbil production is not universal in the genus Allium ( Karpavičienė 2017) and is probably related to the age of respective species and participation of hybridisation followed by polyploidisation. Concerning section Conodoprasum , majority of polyploids including allotetraploid A. telmatum and A. savii do not produce bulbils within the inflorescence ( Brullo et al. 1994, 1997a, 1997b, 1997c, 2008, Bogdanović et al. 2009) while only some polyploid species (e.g., tetraploid A. oporinanthum, Jauzein & Tison 1999 , Tison et al. 2014; tri- to octoploid A. oleraceum, Duchoslav et al. 2013 , Fialová et al. 2014, Fialová & Duchoslav 2014) or even both diploid and triploid cytotypes of A. carinatum Linnaeus (1753: 297) (see Levan 1937, Stearn 1980) and A. melanantherum Pančič (1883: 64) (see Česchmedziev 1979, 1992) produce aerial bulbils.

Allium pseudotelmatum might be confused with some specimens of A. oleraceum due to the presence of bulbils within the inflorescence and dirty white colour of tepals. Considering morphology of bulbils, A. pseudotelmatum differ from A. oleraceum in having bulbils ovate, yellow-greenish tinged with purplish, stipitate (1–3 mm), with long narrow (caudate) apex (1–3 mm) that persist on ripped bulbils ( Fig. 2D ), while A. oleraceum produce usually elliptical bulbils, dirty purplish to brownish, sessile or shortly stipitate (1 mm) ( Fig. 2E ). Moreover, A. oleraceum differ from A. pseudotelmatum in having different leaf shape and structure of epidermal surface ( Fig. 3 , 4 ), longer leaves, campanulate perianth with apparently narrow tepals (1.8–3.2 mm; ratio tepal length:width> 2.2 vs. <2.2 in A. pseudotelmatum ; Fig. 2B, C ) that have variable colour ranging from dirty whitish tinged by green or pink-purplish to more common dirty purplish or brownish, filaments pinkish to purplish, anthers yellow or reddish ( Table 1), and less frequent production of bulblets (0–2) depending on the ploidy level of plant, with smaller size and oval shape, intergrowing the leaf sheaths aboveground ( Fialová & Duchoslav 2014).

To check differences between A. pseudotelmatum and A. oleraceum at molecular level, we sequenced the ITS region, which has become the standard barcode of choice in most investigations for plants ( Fazekas et al. 2012). We recorded only marginal differences between the two A. oleraceum records represented by four ambiguous positions in ITS sequences analysed by Salmeri et al. (2016). On the other hand, substantial differences between A. pseudotelmatum and both A. oleraceum records are displayed by 7 SNP positions and a single nucleotide gap. This observation clearly proved that A. pseudotelmatum and A. oleraceum cannot be considered conspecific.

Presence of bulbils in A. pseudotelmatum may be connected with its odd ploidy level and might be considered as an adaptive mechanism maintaining reproduction ( Abrahamson 1980) because odd-ploidy plants in particular have significant problems with chromosome pairing during meiosis, resulting in generally lowered production of seeds that are usually of low fertility ( Hanelt 1996, Ramsey & Schemske 1998, Jauzein et al. 2002). In addition, A. pseudotelmatum is likely of younger origin (neopolyploid) in comparison with closely related paleopolyploid A. telmatum with an apparently diploidized karyotype ( Bogdanović et al. 2009). We suppose that A. telmatum or progenitors of A. telmatum participated in the origin of A. pseudotelmatum but at the same time we cannot exclude participation of other, presently unknown parental species. Higher ploidy level of A. pseudotelmatum in comparison with A. telmatum may also explain observed larger size of many structures in A. pseudotelmatum , due to frequently reported positive association between the size of cells, organs, or even overall habit and ploidy (e.g., Beaulieu et al. 2008, Balao et al. 2011, Kim et al. 2012). Finally, A. pseudotelmatum represents a rare case of the pentaploid ploidy level observed within the species of the sect. Codonoprasum .

Figures

FIGURE 1. Allium pseudotelmatum. A. Habit. B. Inflorescence. C. Perigon with stamens and anthers. D. Ovary with style. E. Capsule. F. Anther. G. Details of aerial bulbils within the inflorescence. H. Bulb with two daughter bulbs. Length of scale bar represents 1 cm (A, B, C, G, H) or 1 mm (D, E, F). Photographs by Martin DUCHOSLAV.

FIGURE 2. Comparison of important diagnostic characters of Allium pseudotelmatum and A. oleraceum. A. Flower of A. pseudotelmatum. B. Outer and inner tepals of A. pseudotelmatum. C. Outer and inner tepals of Allium oleraceum. D. Mature bulbils of A. pseudotelmatum. E. Mature bulbils of A. oleraceum. Length of scale bar represents 3 mm. Drawn by Martin DUCHOSLAV.

FIGURE 3. A. Leaf cross section of Allium pseudotelmatum. B. Leaf cross section of Allium oleraceum. Anatomical characters of Allium pseudotelmatum: C. Epidermis with well-developed micropapillae, cuticle covers and sunken stoma. D. Detail of palisade tissue and spongy tissue with vascular bundles. E. Colateral closed vascular bundle with sclerenchymatical bundle-sheat around xylem. Length of scale bar represents 500 μm (A, B), 100 μm (D) or 50 μm (C, E). Photographs by Martina OULEHLOVÁ.

FIGURE 4. The arrangement of micropapillae on the surface of leaf epidermis: A. Allium pseudotelmatum. B. Allium oleraceum. Photographs by Martina OULEHLOVÁ.

FIGURE 5. A. Metaphase plate of Allium pseudotelmatum (2n = 40). B. Karyotype of A. pseudotelmatum (2n = 40). Length of scale bar represents 10 μm. Photograph by Aloisie POULÍČKOVÁ.