Abstract
Gaylussacia brachycera (box huckleberry) is a unique relict species and is the monospecific member of Gaylussacia sect. Vitis-idaea. As part of investigations to understand interspecific crossability in Gaylussacia, pollinations were made between evergreen 2x G. brachycera and deciduous 2x G. dumosa (dwarf huckleberry). The primary pollinations succeeded at a low level and produced four viable hybrids. Three of these hybrids had box huckleberry as the female, and one of them, dwarf huckleberry, as the female. Morphologically, these hybrids were distinct from the parents and were generally intermediate. The fertility of the hybrids was low, but one hybrid flowered sufficiently to allow collection of pollen and to backcross it as a male to box huckleberry. Four BC1 hybrids were produced from this cross. The F1 and BC1 hybrids were all verified using simple sequence repeat markers developed for the closely related genus Vaccinium. These hybrids are notable for being the first recorded hybrids of this rare species, G. brachycera, with any other Gaylussacia species, and these hybrids are expected to lead to a better understanding of species relationships both within Gaylussacia and between Gaylussacia and Vaccinium.
Gaylussacia is an Ericaceous genus closely related to Vaccinium. One of the defining characteristics of Gaylussacia is the occurrence of a 10-locular ovary, with each chamber producing a single seed with a hard seedcoat (Kron et al. 2002). By comparison, highbush blueberry, V. corymbosum L., has a five-locular ovary, with each producing variable seed numbers and totaling as many as 50 seed per berry with relatively soft seedcoats (Doi et al. 2018; Nicholson 2011). Based on morphology, Sleumer (1967) organized Gaylussacia into three sections and multiple subsections. Sleumer’s three sections consist of sect. Gaylussacia (encompassing primarily South American species), sect. Decamerium (encompassing most North American species), and sect. Vitis-idaea [comprising only G. brachycera (Michx.) A. Gray]. To date, the most comprehensive molecular investigation of Gaylussacia was that of Floyd (2002), who suggested these sections may not be entirely natural and most notably placed G. dumosa in sect. Gaylussacia. As part of ongoing investigations of crossability both in Gaylussacia and Vaccinium, we conducted crosses with G. brachycera, especially with the idea of developing a more vigorous and fertile derivative that might be useful as a specialty landscape plant.
Three species of Gaylussacia are found natively in southern New Jersey, USA. The two most common species are the black huckleberry (G. baccata Wang.) and the blue huckleberry (G. frondosa L.). Both are common and somewhat generally cohabiting. Their species integrity is maintained by flowering at somewhat different times but also by other incompatibility factors (MK Ehlenfeldt, experimental observation). The third species found in New Jersey is G. dumosa (Andrews) A. Gray, the dwarf huckleberry is relatively rare in New Jersey, and only one genotype was secured for our studies through local acquisition. Sorrie and Weakley (2007) asserted that the species found in New Jersey is more correctly identified as G. bigeloviana (Fernald) Sorrie & Weakley, with the main differences being plant size, hair density, and habitat. Our specimen was a single plant found under conditions more typical of those described for G. dumosa, and we therefore have retained that nomenclature.
Gaylussacia brachycera, the box huckleberry, is a low-growing, rhizomatous, evergreen shrub 10 to 20 cm tall, with distinctly angled stems. It is unique among North American Gaylussacia in having glossy, coriaceous, evergreen leaves lacking resinous trichomes. Its leaves are elliptic, 1 to 2.5 cm × 0.5 to 1.3 cm and are minutely toothed unlike those of other Gaylussacia species. In appearance, they resemble leaves of Japanese Boxwood (Buxus microphylla Siebold & Zucc.), hence its common name. Small pink to white flowers 4 to 5 mm long occur in early spring. The occasional fruit are blue and glaucous, 7 to 10 mm in diameter, and ripen in spring to early summer. G. brachycera is believed to be diploid (2n = 2x = 24), but with some uncertainty (Luteyn et al. 1996). G. brachycera is typically found on wooded hillsides, chiefly in mountainous areas.
G. brachycera is a relatively rare species of Gaylussacia. It has been considered by some to be a glacial relict species, surviving in small vegetatively reproducing colonies, but its origins have alternatively been postulated to arise from multiple waves of North American–South American migration of species (Floyd 2002). It has also been speculated that box huckleberry might be the result of an ancient Vaccinium–Gaylussacia hybridization (Floyd 2002). In either case, its origins remain unclear. G. brachycera was originally identified in a few locations in Pennsylvania (notably near New Bloomfield, PA, USA), but over the course of time, additional colonies have been identified in the United States in Pennsylvania, Delaware, Maryland, West Virginia, Virginia, Tennessee, Kentucky, and North Carolina (Smith and Smith 1971; Wilbur and Bloodworth 2004). The New Bloomfield location has been studied most intensively and was believed to be a single, large, vegetatively spreading clone, originally stretching 400 yards. It has been estimated to be more than 1200 years old based on a calculated annual growth of ∼6 inches (Coville 1919). Coville found that the plants produced few fruit and the few seeds contained therein resulted in weak seedlings that were unlikely to survive in the wild. Subsequent molecular studies have shown the New Bloomfield colony (denoted as Baird) to be composed of two genotypes, but with such a minor variation as to suggest a somatic mutation of the founder clone (Pooler et al. 2008). A different Pennsylvania colony (WardB) was found to consist of three genotypes, and its origin has been suggested to be the result of several founder clones growing with little interclonal competition (Pooler et al. 2008). Other studies have documented genetic diversity between clones collected across a wider region (Pooler et al. 2006).
G. dumosa, the dwarf huckleberry, is a plant of moderate stature, up to 40 cm tall, that is rhizomatous and forms open colonies. Leaves are coriaceous, oblanceolate to elliptic, and 1.4-4 mm × 0.6-2.3 mm with glandular pubescence on both surfaces. New foliage is glossy with prominently reticulated venation and texture. Flowers are produced in racemes 3 to 7 cm long, having 4 to 10 flowers. Flowers are white, urceolate to campanulate, 5 to 10 mm long, and approximately equal in diameter. During initial development, corollas are distinctly pleated. The fruit (drupe) is black, glandular-pubescent, 6 to 11 mm in diameter. G. dumosa ranges from Newfoundland, Canada, to Florida and Louisiana in the United States (Luteyn et al. 1996; Nicholson 2011). G. dumosa is deciduous under New Jersey conditions. Unlike G. brachycera, G. dumosa, in our observations, possesses good fertility and reproduces in a normal sexual manner.
The crosses reported herein began as a project at the instigation of Dr. Margaret Pooler of the US Department of Agriculture–Agricultural Research Service Floral and Nursery Crops Laboratory (Beltsville, MD). Dr. Pooler, Ruth Dix, and Dr. Robert Griesbach (also of the Floral and Nursery Crops Laboratory) had collected and/or acquired a number of G. brachycera accessions and hoped to breed selected parents and develop improved genotypes for potential use as an ornamental groundcover (Pooler and Olsen 2006). As noted, G. brachycera has been historically recognized to have extremely low fertility (Coville 1919), but a few modestly fertile genotypes existed among the collected specimens (Pooler M, personal communication). No crossability of G. brachycera with any other Gaylussacia spp. has been reported.
The glossy leaves and larger ornate flowers of G. dumosa were considered desirable traits to introgress into G. brachycera. It was also believed that G. dumosa might introduce greater vigor and improved fertility. Our objective was to make an attractive and fertile hybrid that was three-quarters G. brachycera and one-quarter G. dumosa, recovering most of the G. brachycera phenotype, especially the evergreen habit.
Materials and Methods
Plant material.
Gaylussacia genotypes used in these experiments are presented in Table 1.
Gaylussacia genotypes used in experiment.
Crossing protocols.
For all plant material, pollen was extracted from open flowers by manual manipulation and collected on glassine weighing paper. If pollen was needed for longer term work, it was stored for up to a month under refrigerated, desiccated conditions.
To perform pollinations, a graphite pencil tip was dipped into the collected pollen and then used to apply the pollen to the stigmas of unemasculated flowers in an insect-free greenhouse. Pollinations were made on what were judged to be mature stigmas. All pollinations were performed in an insect-free greenhouse, and because it was expected that hybrids would be morphologically recognizable, the female cultivar parents were not emasculated.
Ploidy determinations.
Given that some previous crosses within Ericaceae had produced anomalous triploids and tetraploids (Ehlenfeldt et al. 2023), the ploidy of parents and F1 hybrids was verified using flow cytometry. Ploidy determinations were performed as described by Ehlenfeldt and Luteyn (2021) using the following procedures. Sampled leaf material (1 cm2/20 to 50 mg) together with leaf material of an internal standard with known DNA content (Zea mays L.) was chopped with a sharp razor blade in 500 mL of extraction buffer (CyStain PI absolute P buffer, catalog number 05 to 5502; Partec, Münster, Germany) containing RNase, 0.1% dithiothreitol (DTT), and 1% polyvinylpyrrolidone (ice cold) in a plastic petri dish. After 30 to 60 s of incubation, 2.0 mL staining buffer (CyStain PI absolute P buffer) containing propidium iodide (PI) as fluorescent dye, RNA-se, 0.1% DTT, and 1% polyvinylpyrrolidone was added. The sample containing cell constituents and large tissue remnants, along with the internal standard, were then filtered through a 50-mm mesh nylon filter. After an incubation of at least 30 min at room temperature, the filtered solution with stained nuclei was analyzed with the flow cytometer [CyFlow ML (Partec) with a green diode laser 50 mW/532 nm (for use with PI); software: Flomax Version 2.4 d (Partec)]. The DNA quantity of the unknown samples was calculated by multiplying the DNA quantity of the internal standard by the DNA ratio of the relative DNA quantity of the unknown sample and the internal standard. Calculated DNA quantities were compared with a set of standards covering a diploid to hexaploid range (2x V. darrowii ‘Fla 4B’, 4x V. corymbosum cv. Duke, and 6x V. virgatum cv. Powderblue) to determine basic ploidy levels.
Male fertility.
Pollen samples were stained with acetocarmine jelly (75% acetic acid with iron acetate) and prepared according to the recipe of Jensen (1962). Pollen samples were assayed for quantity, stainability, and general condition. For evaluating general pollen condition, our ratings were as follows: very good = almost all tetrads, good = tetrads and triads, fair = almost exclusively triads, and poor = mostly aborted grains.
Female fertility.
The number of flowers pollinated to evaluate female fertility varied depending on flower availability. Pollinations and fruit set were recorded. Fruit was collected when ripe and measured for fruit size (diameter in millimeters) at the time of seed extraction. Extraction was performed manually under a dissecting microscope and the seed were counted and rated for quality. Seed were classified as good (g), good/fair (g/f), fair (f), fair/poor (f/p), poor (p), or aborted. ‘Good’ and ‘fair’ described seed that subjectively ranged from those considered fully normal to those somewhat reduced in size and/or development but nonetheless were judged likely to be capable of germination. ‘Poor’ described seed that displayed reduced size and/or development, often flattened or brown, and judged less likely to be capable of germination. ‘Aborted seed’ were those that were flat and brown and generally translucent. Notes were made of the size of aborted seed. For purposes of this work, we report only seed totals that combine categories of good and good/fair.
Seed were sown directly from the fruit on a greenhouse mist bench in a soil mix composed of a 50:50, peat:sand mixture. Seed that did not germinate in 10 weeks were given a cold treatment of ∼1000 h at 3 °C, then returned to the mist bench for further germination attempt. At approximately a three true-leaf stage, seedlings were transplanted to 36-cell flats. All primary hybrids were transferred to 3-L pots in their second season.
Plant material and DNA isolation.
Parent plants and hybrid seedlings were grown in a tunnel greenhouse at the P.E. Marucci Center for Blueberry & Cranberry Research in Chatsworth, NJ, USA in 2023. Fresh cuttings were sent by overnight mail to the USDA Cranberry Genetics and Genomics Laboratory (CGGL) in Madison, WI, USA for genetic testing. Upon arrival to CGGL, plant tissue was processed by using a sentry 2.0 BenchTop lyophilizer (VirTis, Gardiner, NY, USA) and DNA was extracted from lyophilized samples.
DNA was extracted from 0.03-0.04 g of freeze-dried leaf tissue per sample using a modified hexadecyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1987) with beta-mercaptoethanol (2 µL in 750 µL CTAB) and incubated at 65 °C for 1 h. Solubilized DNA from each sample was retrieved from the aqueous layer after adding chloroform:isoamyl alcohol (24:1) and centrifugating at 14,000 gn for 8 min. DNA was precipitated from the aqueous layers by adding cold isopropanol, placing in a freezer at –20 °C overnight, then centrifugating at 14,000 gn for 25 min to pellet the DNA. The DNA pellet was washed twice in cold 70% ethanol, then resuspended in 50 µL of 1× TE buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA pH 8.0) with 3 µL of RNase-A. The DNA in 1× TE with added RNase-A was incubated at 37 °C for 3 h to remove any RNA. DNA was stored at 4 °C until use.
DNA amplification, fragment analysis, and validation of simple sequence repeat polymorphisms.
Polymerase chain reactions (PCRs) for each plant sample were assembled in duplicate, each reaction being a total volume of 8 µL. Individual reactions comprised 5 µL 1× JumpStart REDTaq ReadyMix (Sigma, St. Louis, MO, USA), 1.0 µL containing 10 to 20 ng of DNA per sample in 1× TE buffer, 0.5 µL of 5M Betaine PCR reagent (Millipore Sigma, MA, USA), 0.5 µL of 5 µM hexachloro-6-carboxy-fluorescein (HEX)-M13 primer, 0.5 µL of 5 µM forward simple sequence repeat (SSR) primer appended with the M13 5′CACGTTGTAAAACGAC-3′ sequence, and 0.5 µL of 50 µM reverse SSR primer appended with 5′-GTTTCTT-3′. The modifications to the SSR forward (Schuelke 2000) and reverse (Brownstein et al. 1996) primers serve to facilitate fluorescent labeling of PCR fragments and promote nontemplated adenylation, respectively.
PCR was completed on S1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) programmed for a single melting step at 94 °C for 3 min, followed by 33 cycles of 94 °C, 55 °C, and 72 °C for 15 s, 90 s, and 2 min, respectively, and a final extension at 72 °C for 30 min.
To analyze the PCR fragments, 1 µL of the HEX-labeled PCR product was added to a 10 µL mix of formamide and Custom MapMarker ROX 75 to 375 bp carboxy-X-rhodamine ladder (BioVentures, Murfreesboro, TN, USA) at a ratio of 1000 µL to 25 µL, formamide to ladder. The fragment-formamide mixes were sent to Functional Biosciences, Inc. in Madison, WI, USA for fragment analysis. Samples were run on an ABI 3730 DNA Analyzer (Applied Biosystems, Waltham, MA, USA) fluorescent sequencer with a 50-cm capillary array. The raw data were returned to CGGL, and SSR fragment sizes (in bps) were determined using the GeneMarker software version 1.91 (Soft-Genetics LLC, State College, PA, USA).
The SSR primers for this study were selected from a validated polymorphic SSR marker library developed for Vaccinium macrocarpon (Schlautman et al. 2015). A subset of this SSR marker library was shown to be cross-transferable to many other Vaccinium species (Rodriguez-Bonilla et al. 2019). A total of four SSR primers were identified that amplified polymorphic loci in the parents with expected segregation in the offspring.
The sequences of three primers in the SSR panel (409500_K63, 281884_K70, 172672_K70) are reported in Rodriguez-Bonilla et al. (2019). The last SSR primer (6ms4e4b) was tested in an unpublished dataset from Rodriquez-Bonilla. The forward primer sequence for “6ms4e4b” is 5′-CACGACGTTGTAAAACGACGGCCAAGGTTCTACCCTTTC-3′ and the reverse primer is 5′-GTTTCTTCAACTACCCACCACCACCAT-3′. The first 19 bases in the forward primer, starting from the 5′ end, are the M13 sequence (underlined), and the first 7 bases in the reverse primer, starting from the 5′ end, constitute the primer modification to promote adenylation, also known as PIG-tailing.
Results
Production of F1 and BC1 hybrids.
Each fruit of Gaylussacia is expected to bear 10 seeds in the case of fully successful crosses. In intraspecific pollinations, these seed are typically pale with a hard seedcoat. Across all the G. brachycera × G. dumosa hybridizations, seed quality was found to be quite variable, ranging from “very good” and fairly typical, to seed that was considered underdeveloped and green. The relative success rates in producing viable hybrids suggests that only seed rated as “very good” or “good” succeeded in germinating and producing plants (Table 2). For the crosses of ‘Berried Treasure’™ × G. dumosa US 1875, only five fruit were set from 45 pollinations (11%) with an average of 9.4 seeds/fruit (47 seed produced). For crosses of G. brachycera US 1989 × US 1875 G. dumosa, only two fruit were set from 50 pollinations (4%), with an average of 7 s/f (14 seed produced). Across all pollinations fruit set was 7% and seed production was 8.7 seeds/fruit.
Table 2. Primary and backcross hybrids of Gaylussacia brachycera and G. dumosa.
Among reciprocal crosses, only crosses of US 1875 (G. dumosa) × NA 71555_5 BX (G. brachycera) yielded viable seed. This combination produced US 1981. The metrics of this specific cross are not available, but for comparison, a total of 56 hybridizations of US 1875 (G. dumosa) with the G. brachycera genotypes ‘Berried Treasure’™ and US 1989 produced no fruit or seed.
In 2021, the F1 hybrid US 2520-A flowered profusely (Fig. 1C) and although shed was low, there was a sufficient quantity of flowers to collect small amounts of pollen for use in crosses with G. brachycera females. Only crosses using ‘Berried Treasure’™ as a female succeeded (Table 2). The original collection notes for ‘Berried Treasure’™ indicate that it successfully produced fruit without cross-pollination; thus, it may have better female fertility than the other clones. These crosses succeeded at a rate of four fruit per 28 pollinations (14.2%). These four fruit produced 24 seeds rated as “good” and 14 as “good-fair.” Using the total of these two classes as the realized seed potential, the crosses produced 9.5 s/f. From these, four viable seedling genotypes were produced. These seedlings have the expected composition three-quarter G. brachycera and one-quarter G. dumosa. Initial morphological observations of these seedlings suggest a mature phenotype that will be approaching G. brachycera and a likelihood of evergreen physiology (Fig. 1D). No observations have yet been made of flowering or fertility.
Flow cytometry.
Previous interspecific crosses within Ericaceae have sometimes produced unexpected triploids and tetraploids (Ehlenfeldt and Ballington 2017). Therefore, the ploidy of all parents and F1 hybrids was verified by flow cytometry. All the parents and hybrids were found to be diploids (2n = 2x = 24) compared with the 2x V. darrowii ‘Fla 4B’ (sect. Cyanococcus) standard (1.16 pg). The four species parents had values of: 1.01 pg (G. brachycera NA 71555_5 BX), 1.01 pg (G. brachycera ‘Berried Treasure’TM), 1.04 pg (G. brachycera US 1989), and 1.10 pg (G. dumosa US 1875). Values for the four interspecific hybrids were 1.08 pg (US 1981), 1.11 pg (US 2511), 1.04 pg (US 2520-A), and 1.11 pg (US 2520-B).
SSR fragment analysis and F1 hybrid validation.
SSR fragment sizes (in bps) amplified by PCR for the Gaylussacia parents are shown in Table 3. Four SSR primers from Rodriquez-Bonilla et al. (2019) (409500_K63, 281884_K70, 172672_K70, 6ms4e4b) showed useful polymorphic variation within and across the Gaylussacia species in this study and were used to confirm the parentage of putative Gaylussacia hybrids.
Simple sequence repeat (SRR) alleles (in bps) of Gaylussacia parents, F1 hybrids, and backcrosses.
The presence of two alleles per hybrid progeny, one from each of the two parents, was seen in most of the hybrids. For example, for primer 409500_K63, US 2520-A inherited the 347 bp SSR allele from US 1875, whereas US 2520-B inherited the 349 bp allele from US 1875 (Table 3). Our SSR data validates hybrid origin of all four F1 hybrids: US 1981 (=US 1875 × US 1989), US 2520-A (=US 1989 × US 1875), US 2520-B (=US 1989 × US 1875), and US 2511 (BT × US 1875) (Table 3).
The progeny of US 1989 × US 1875 showed expected inheritance of SSR loci across all four primers. US 2520-A inherited one allele from G. brachycera ‘US 1989’ and a second allele from G. dumosa ‘US 1875’ for all four primers tested. In the case of its sibling, US 2520-B, three of the four SSRs (409500_K63, 281884_K70, 6ms4e4b) show the same inheritance behavior, with the last marker (172672_K70) only showing one of the two alleles expected, suggesting a dropped allele (Dewoody et al. 2006; Ehlenfeldt et al. 2024).
Similar to F1 progeny, all four backcross (BC1) progeny of ‘Berried Treasure’™ × US 2520-A showed clear inheritance of alleles from both parents for primers 409500_K63 and 172672_K70 (Table 3). Primer 281884_K70 amplified on a single allele. The 311 bp allele from primer 281884_K70 is likely homozygous in each parent. For primer 6ms4e4b, BC1 hybrids A, C, and D also show clear inheritance of an allele from the ‘Berried Treasure’™ progenitor and the F1 US 2520-A, but hybrid B has a dropped allele of 192. Taken altogether, the molecular data validate the BC1 Gaylussacia hybrid progeny in this study.
F1 hybrid morphology.
Hybrids were distinctive in appearance from either parent and were generally intermediate but showed considerably greater influence of G. dumosa, especially in plant stature (Fig. 1A and B). The hybrids US 1981, US 2520-A, and US 2520-B were similar in stature averaging 20 cm in height (range 15 to 23 cm), whereas US 2511 was a more upright plant at 35 cm in height. A general description of hybrid morphology pertains: The hybrids possess stems that are initially broadly angular but become terete as they mature. The stems are densely covered in white, simple, nonglandular hairs. Axillary buds are small and inconspicuous. Leaves are coriaceous (but less so than G. dumosa), dark green on the upper surface and lighter whitish-green beneath, generally elliptic, and intermediate in size to the parents, with a broadly acute to rounded apex, and appearing to lack a distinct terminal mucro. Leaves are essentially glabrous; margins are flat, slightly thickened, crenate, and adorned with teeth, each tipped with a deciduous reddish glandular hair. Leaf venation is pinnate, with three to four inconspicuous lateral veins. The midrib and lateral veins are slightly impressed on the upper surface. Notably, there are no resinous hairs present on the hybrids.
Inflorescences composed of five to six flowers; flowers white, tinted with pink on petal bases, ridges, and on sepal tips during initial phases; pink coloration fading as flowers mature; corollas spherical (7 mm × 7 mm) to slightly cylindrical (7 mm × 8 mm); corollas slightly ribbed (Fig. 2).
Hybrids were reasonably vigorous but not fast growing. They were moderately susceptible to powdery mildew in the greenhouse, but it is our experience that young plants of exotic hybrids often express susceptibility to powdery mildew but may be less obviously susceptible under field conditions and may also become less susceptible as they emerge from juvenility.
The hybrids retained their foliage through winter when maintained under cold but nonfreezing conditions, but they shed previous-season foliage during the following spring growth period. Thus they cannot be considered evergreen.
Due to limited flowering, the hybrids were evaluated exclusively as potential pollen sources. Only US 2520-A bloomed sufficiently in 2021 to be used as a pollen parent. Female fertility was not assessed but was assumed to be similarly low.
Pollen fertility of F1 hybrids and parents.
As might be expected from wide interspecific hybrids, pollen development was adversely affected, with problems ranging from a total lack of pollen shed in the worst case to low percentages of perfect tetrads in the best case.
US 1981—pollen nonexistent.
US 2511—very light pollen shed; a few well-formed tetrads, others with one to two viable microspores (Fig. 3).
US 2520-A—Pollen was not microscopically examined, but likely to be similar to US 2520-B. A low level of male fertility was inferred from its succeeding as a parent in BC1 crosses (see Production of BC1 Hybrids).
US 2520-B—very low shed; no perfect tetrads, but some tetrads with 1 to 2 viable microspores.
Pollen samples of the parent genotypes were also examined for comparison purposes.
Although the three G. brachycera clones all showed reasonable pollen shed, pollen development irregularities noted were considered a reflection of the historically clonal reproductive mode of this species. G. dumosa, as a more conventionally sexual species, exhibited both good shed and a high level of well-formed tetrads.
G. brachycera NA 71555_5 BX—relatively good shed; some perfect tetrads; many tetrads with three viable microspores/one aborted.
G. brachycera ‘Berried Treasure’™—relatively good shed; high frequency of perfect tetrads, but some displaying shape distortion.
G. brachycera US 1989—relatively good shed; ∼15% perfect tetrads, 50% mixed tetrads with one, two, or three viable microspores, and 35% aborted (i.e., no viable microspores).
G. dumosa US 1875—very good shed; high frequency of perfect tetrads; lesser quantity of triads.
Discussion
The F1 G. brachycera–G. dumosa hybrids are the first recorded hybrids of G. brachycera with any other Gaylussacia species and are notable for being not a single rare hybrid but multiple hybrids having G. brachycera as both the female and male parent. This is in contrast to some other rare interspecific hybrids only produced unidirectionally in genus Vaccinium (Covarrubias-Pazaran 2016; Rodriguez-Bonilla 2019). The hybrids have been relatively slow growing, but G. brachycera itself is quite slow in its development and spread (Coville 1919).
G. brachycera belongs to sect. Vitis-idaea, and G. dumosa to sect. Gaylussacia. These primary hybrids are therefore intersectional hybrids within Gaylussacia. In Vaccinium, the fertility and compatibility of intersectional hybrids can vary depending upon relative relatedness of the parent species, and the ploidy of the resultant hybrids (Edger et al. 2022). Although tetraploid hybrids almost always exhibit some fertility due to allosyndesis (Clausen and Goodspeed 1925), fertility of diploid Ericaeous hybrids varies (Christ 1977; Vorsa et al. 2009; Zeldin and McCown 1997). By most assessments, G. brachycera is significantly diverged from the broad spectrum of Gaylussacia species, being both morphologically divergent and possibly a relict species (Floyd 2002). Thus, the low fertility observed in these hybrids is not surprising considering the level of apparent unrelatedness between the two parents (Floyd 2002), and the lack of fertility and the documented clonal reproduction of G. brachycera (Coville 1919). Nonetheless, at least one of our hybrids produced viable BC1 hybrids at a very low frequency. The Gaylussacia F1 hybrids and BC1 hybrids were verified by SSR marker analysis (Table 3).
On the basis of fruit chemistry characteristics, Forney et al. (2012) suggested that one of our parent species, G. dumosa, might be more closely aligned with the genus Vaccinium than with Gaylussacia, despite its characteristic 10-locular/seeded Gaylussacia morphology. This suggestion raises the possibility that our hybrids could be even rarer intergeneric hybrids. However, in previous work, we readily generated numerous fertile hybrids of G. dumosa × G. baccata Wang., thus supporting the taxonomic status of G. dumosa as a true huckleberry and not a Vaccinium (data not shown). The pollen fertility of our G. brachycera–G. dumosa hybrids although low, also supports this conclusion.
As part of the development of a crossability “roadmap” for Vaccinium and other Ericaceae, we have pursued investigations of crossability into the nearest related genus, Gaylussacia. In this article, we have shown that like Vaccinium, divergent Gaylussacia species have the potential for reproductive compatibility despite significant morphological differences and the evolution of divergent environmental adaptations. In our investigations, there appear to be greater hybridization barriers between the two main sympatric Gaylussacia species within our New Jersey locality, G. baccata and G. frondosa (both in sect. Decamerium) (Floyd 2002), than between G. brachycera and G. dumosa. We believe the hybridization of G. brachycera and G. dumosa will lead to a further understanding of the relatedness and crossability possibilities between Gaylussacia and Vaccinium.
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