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Molecular Characterization of Wild Mushrooms: A Paradigm Shift from Morphotyping

  • Madhusmita Borthakur
  • S. R. JoshiEmail author
Chapter
Part of the Fungal Biology book series (FUNGBIO)

Abstract

The traditional morphological study for the classification and identification of higher organisms depends on the morphotypic features. Development of precise molecular markers and techniques along with statistical tools have aided in identification process to authenticate the existing and widely used morphometry approaches. Significant to the development of barcodes based on DNA sequences in higher eukaryotes which include fungi like mushrooms, marker based molecular approaches have become more reliable and easily usable tools. The present chapter presents a brief overview of the mycological shift from phenotypic to molecular taxonomy. Exploration of various protein encoding and mitochondrial cytochromeoxidase-I barcodes in animals has opened their usage in few higher fungi but due to the presence of intron and lack in resolution has prevented their usage as fungal universal barcode. As proposed by the Consortium for the Barcode of Life in the year 2012, the fungal nuclear ribosomal internal transcribed spacer is considered to be the universal fungal barcode due to its hypervariable and tandem repeated copy number added by its easy amplifiability even with trace amount of nucleic acid.

Keywords

Mushrooms Identification Molecular markers DNA barcode 

3.1 Introduction

Being close to nature, ethnic communities have proximate association with natural resources and mushrooms are one resource that has been harvested and used by mankind since time immemorial. This has resulted in acquisition of immense knowledge about the benefits and utilization of different bioresources for consumption and medicinal purposes (Sawian et al. 2007; Singh et al. 2012). In the Asian sub-continent, mycophilic societies are associated among the indigenous people of North Eastern India, Western Ghats and North Western India, China. The local inhabitants are often seen collecting mushrooms from their neighboring localities, meadows and forests for consumption and for selling them to earn partial livelihood for their family during monsoon season when other forest products are unavailable for harvest.

Ethnomycological knowledge among Indian tribal population has been profound and broad with about 283 wild types of mushrooms being consumed out of 2000 edible species which are recorded worldwide (Purkayastha and Chandra 1985). These mushrooms which are often consumed by the ethnic community are harvested and picked from the forest area based on the traditional knowledge about their morphology and the toxicity. This knowledge on the edibility and the medicinal importance of the mushrooms has been passed from one generation to the other in the form of word of mouth without much record and scientific documentation.

The primary identification of mushroom involves its phenotypic characteristics and its palynological study. The common morphology of a mushroom is its umbrella shape structure but few resemble toads sitting atop on the cap. The common oyster mushroom and the family of Amanita possess distinct gills which are important for its identification. Genus Russula is known to possess hollow white stalk while Boletus consists of a bulbous base at its stalk considered as important features for their identification. The dimension and the colour of the spores (when stained with Lactophenol cotton blue and Melzer staining ) are also an important observable features in identification of mushrooms (Figs. 3.1a, 3.1b and 3.1c) Since a single spore is almost invisible to naked eye, spore prints in a dark or lighter background have often been considered for identification purposes. The colour of the spore prints varies during different growth stages of a mushroom. They are mostly whitish to creamish during younger stage to brownish lilac at maturity. The preliminary identification of mushrooms is solely based on morphological features during field observation and microscopic features of its spores, pileus, stipe, volva (if present) and host. Few mushroom specimens are described and identified till the genus level solely based on the morphotypic features (Table 3.1). The palynological parameters can also be considered as important tool for their identification (Table 3.2). Although morphological and microscopic features have been the basis for identification of mushrooms, there are many unsolved dilemma left in the typing and classification at lower taxon level because of the paucity of morphological features (Hibbett et al. 2007). This is further complicated by other factors which include the convergent evolution and cryptic speciation.
Fig. 3.1a

Morphotyping and photomicrographical analysis of mushrooms of Meghalaya, India using Lactophenol cotton blue and Melzer staining (100xmagnifications)

Fig. 3.1b

Morphotyping and photomicrographical analysis of mushrooms of Meghalaya, India using Lactophenol cotton blue and Melzer staining (100xmagnifications)

Fig. 3.1c

Morphotyping and photomicrographical analysis of mushrooms of Meghalaya, India using Lactophenol cotton blue and Melzer staining (100xmagnifications)

Table 3.1

Morphological characterization of few mushrooms of Meghalaya, India

Specimen identity number

Fruiting body (Pileus)

Stalk (Stipe)

Gills (Lamellae)

Morphological identification

MBSRJ1

5-11 cm wide;Perennial asci shaped with wavy lobed edges, yellow with white margin, leathery tough to velvety in texture

Absent, the asci shaped cap attach to bark of the tree

The pores are mostly small whitish to tan in colour

Formitopsissp.

Order: Polyporales

Family: Formitopsidaceae

MBSRJ2

4.5–8.3 cm wide, convex with margin tucked when young but broadly convex to flat at maturity, pale brownish yellow

3–8.1 cm long,0.8–2.2 cm thick, white hollow dry in texture nearly tapering base

Close distinct gills attached to the stalk pale yellowish to white

Russulafoetens

Order: Russulales

Family: Russulaceae

MBSRJ6

1-5 cm wide, conical to bell shapedwith a central bump, inrolled margin with radial lined, grayish to dirty tan in colour with a dark brown center, bald tacky texture

4.3–7.8cmn long1-2 cm thick, hollow, bald, slender with few tiny fiber like filaments colour varies from white to tan yellowish

Whitish attached to the stalk, prominentpinkish to brownish cross vein at maturity

Mycenagalericulata

Order: Agaricales

Family: Mycenaceae

MBSRJ8

2-5 cm convex wide to nearly flat, dry, velvety in touch, yellowishwith a darker pointed center, the margin sometimes inrolled to wavyin nature

2.5–8.5 cm long, 3.5–6.5 cm thick, tapering at the base, bright brownish to rusty brown from the upward base, faint ring like zone at the end of the stalk

Close crowded often attached to the stalk yellowish to olive yellow in colour, short gills are often connected to one another

Hypholomafasciculare

Order: Agaricales

Family: Strophariaceae

MBSRJ11

Upto 3.5 cm wide, irregularbracket shaped, flattened convexthick leathery, with marginal zone of whitish to pale lilac in colour

Absent, cap attached to tree bark

Pores are angular to erodic spines atmaturity lilac tobrownish spores are often seen at the upper surface of the pores

Trichaptumbioforme

Order:Polyporales

Family:Polyporaceae

MBSRJ12

4.5–8.5 cm wide, broadly convex,with a hollow depression continues to the stalk, dry fuzzy wrinkled purplish to purplish brown, the margin is inrolled wavy, purplishto brownish in colour

6 cm long and 1.5 cm wide, slightly broaden towards the end, colour similar to the cap, presence of tiny hairs at the basalend

Frequent short closer gills continuing to the stalk whitish to pale brownish in colour

Panussp.

Order: Polyporales

Family: Panaceae

MBSRJ13

1.5-3 cm wide, convex to nearly flat at maturity, slightly bell shaped sticky moist to tacky in touch, yellowish to brownish orange in colour, margin inrolled finely lined

2–5.4 cm long, brownish to reddish brown at the basal stalk, dry to moist at maturity

Gills attached to the stalk short frequent close or nearly distant, gills turn into rusty brown atmaturity

Galerina sp.

Order: Agaricales

Family: Hymenogastraceae

MBSRJ18

9.5 cm wide, broadly convex, shallowly depressed, continuing the stalk, wavy edges, soft whitish incolour

7.5 cm long, white with a tapering end, solid

Runs down the stem, crowded, forking, pale creamy

Lactariusglaucescens

order: Agaricomycetes

Family: Russulaceae

MBSRJ26

5.5 cm wide, convex to broadlyflattened, fairly smooth to slight depression at the center, often seendarker at the center, the margin is peeled off to slightly lined at maturity

5.5 cm long, white hollow, 2.5 cm thick, firm, fairly smoothen in touch

Gills attached running slightly down to the stalk whitish to creamish with frequently closer to each other

Russulalepida

Order: Russulales

Family: Russulaceae

MBSRJ35

17.8 cm wide, convex, broadly flattened, greasy to tacky, browncap, margin lobed, wavy, irregular with thick white flesh and brownish fruiting body

9.5 cm long, 5.5 cm thick, broaden at the base of the stalk, pale yellowish irregular stalk tapering at the base

Pores are stuffed long cylindrical with regular dimension, yellowish at the surface. Pores are seen attached to the stalk.

Boletus sp.

Order: Boletales

Family: Boletaceae

MBSRJ37

7.7 cm wide, round to convex,shallowly depressed at the center, dry smooth to touch,purplish pink outer layer, the margin slightly lined to inward rolled

8.4 cm long to 2.2 cm thick, whitish, hollow, brittle to dry in touch

Gills slightly running down the stem, closed to crowded near the stem, whitish in colour to spotted brown near the stalk, soften in touch

Russulasp.

Order: Russualales

Family: Russulaceae

MBSRJ38

2.5–3.9 cm wide, asci with wavy lobed inrolled margin, creamishto dark brown, surface slightlyumbonated, sticky, moist, smooth fibrillose in texture

Stipe is absent

Lamellae sinuate, crowded, whitish to pale yellowish at the time of maturity

Campanophyllumproboscideum

Order: Agaricales

Family: Cyphellaceae

MBSRJ39

7.3 cm wide, white to creamishin colour, funnel shaped withcap continuing to the stalk, white firm flesh crumbly fruiting body with the presence of milky latex like substance on breakage

6.4 cm long hollow stalk, whitish in colour

Gills are decurent and narrow and distantly present.

Lactifluusvellereus

Order: Russulales

Family: Russulaceae

Milky white latex like substance are often seen on breakage

MBSRJ45

Elongated spike like with a capdiameter of 6 cm, fragile, rough to pitted and ridge, olive brown dark brown in colour

Stipe hollow elongated withlength range from 7.9–10.7 cm,thick with the presencepotholes, presence of a lateralpale brown volval remnantaround the stalk base

Phallus sp.

Order: Phallales

Family: Phallaceae

MBSRJ48

1.8–3.9 cm across, broadly convex to bell shaped, with a central depression, soften in touch, dry, pale brown with the surface wrinkled or ribbed

1.7–2.9 cm long, equal, dry, presence of fine hair, pale at the apex, reddish brown to blackish at the stalk base

Gills broadly attached to the stalk, buff to whitish in colour, presence of collar that attach the gills to stalk

Micromphalefoetidum

Order: Agaricales

Family: Marasmiaceae

MBSRJOY

Upto 12 cm wide, broadly convexto flat at maturity, kidney shape with inrolledmargin,whitish in colour

Stalk is absent or lateral in few, 1 cm long, whitish, soften in touch

Gills continuing the lateral stalk, whitish to creamish depends on age with spores at upper layer

Pleurotusostreatus

Order: Agaricales

Family: Pleurotaceae

MBSRJ51

6.2 cm wide, broadly convex to flat,dry to sticky, yellowish to dark brownish in colour

6 cm long, slender, equal,tough, with enlarge base dark brown to blackish in color

Yellowish to orange pores, depressed at the center of stalk, uniform tube size

Boletus pseudocalopus

Order: Boletales

Family: Boletaceae

MBSRJ54

12.1 cm wide, stipitate, funnelshape, colour varies formingtwo zone, the inner darker yellow to brownish to outerlighter to pale yellowish to whitish, edges wavy, the cap continues the stalk

Stalk 4.8 cm long, tough, hollow, darker zone just beneath the cap

The pores are yellowish, small uniform

Amaurodermarugosum

Order: Polyporales

Family: Ganodermataceae

MBSRJ55

13.5 cm wide, broadly convex with a slight central bump, cap surface covered with dark brown scaly fibers over beige surface with dark center

7.8 cm long with 1 cm wide, hairy surface, ragged, white ring with brown scales undersurface, the colour similar to the cap

Gills are crowded, whitish, few are scaly similar to cap

Echinodermaaspera

Order: Agaricales

Family: Agaricaceae

MBSRJ57

6.5 cm wide, convex, wavy edges, moist surface, withyellowish flesh, the cap colour is yellowish brown

The stalk is wavy with a tapering end, fibrous surface, 6 cm long, reddish brown surface

Fine angular pores, yellowish, long, decurrent, coarse, moist

Suillussp.

Order: Boletales

Family: Suillaceae

MBSRJ58

5.5 cm wide, broadly flat with a depressed center,yellowish with striated edges

White hollow stalk, 3.5 cm long, equal attach from the cap center

White decurent gills, forked at the stalk

Gymnomyces sp.

Order: Russulales

Family: Russulaceae

MBSRJ59

16.5 cm wide, wavy edges,broadly convex, dry surfacerugulose, faded brownishyellow

7 cm long, 5 cm wide, apex tapering, dark brown, fibrous scales at the stalk, apically sub reticulate

Long slender pores, yellowish, abundant, close, depressed at the stipe

Leccinumrugosiceps

Order: Boletales

Family: Boletaceae

MBSRJ61

5.5 cm wide, convex,margininvolute, black tough scalous fibers on beige surface

3 cm long with tapering end, hairy fibrous surface similar to the pileus

Whitish to grayish pores underneath the cap

Strobilomycesfloccopus

Order: Boletales

Family: Boletaceae

MBSRJ62

Wood decaying with a capdiameter of 7.5 cm, brown toblackish asci, wavy margin,tough surface,cap continuingthe attached stalk

Thin slender stalk with a tapering end, colour similar to the fruiting cap

Highly polyporous attached undersurface the cap, pale yellowish to white

Amauroderma sp.

Order: Polyporales

Family: Ganodermataceae

MBSRJ63

3.5 cm convex cap, smooth, moist, reddish brown, sticky,remains of partial vein on the edges

Pale yellowish hollow stalk of 3.5 cm length, 1 cm wide attach to the cap, soft smooth surface

Pale yellowish, attached to stem, nearly distant, close with a vein like structure

Agrocybeochracea

Order: Agaricales

Family:Agaricomycetes

MBSRJ64

7.25 cm convex flat with acentral depression, sticky surface, the margin lined,bruising green to beige, bald

6.5 cm long, hollow,pitted somewhat, color similar to cap

Gills continues to stalk, nearly distant, spotting green in colour

Lactariuspurpureus

Order: Russulales

Family: Russulaceae

MBSRJGN

15 cm wide, kidney shaped, wavy, rough surface, cracked, sometimes velvety, brownish with white margin, attached to tree bark

Stalk absent

Highly polyporous, tough, circular creamish to yellowish pores

Heterobasidionannosum

Order: Russulales

Family: Bondarzewiaceae

MBSRJ67

Broadly convex 3.5 cm, withshallow depressed center, dry, brownish, Somewhat wrinkled with lined margin with radial splits

5 cm long, equal withslight flaring at the end velvety to fine hairy in touch, brownish to nearly blackish at the base

Gills whitish to yellowish distantly present, attached to stalk

Gymnopussubnudus

Order: Agaricales

Family: Marasmiaceae

MBSRJ69

Cap wide of 5 cm, brownish, tough with shallow depressed at the center, curvy edges

Short stalk of 2.5 cm long, colour similar to the cap

Gills attached to stem, distantly apart, adnate, creamish in colour

Collybia sp.

Order: Agaricales

Family: Tricholomataceae

MBSRJ70

Convex tongue shape withcap diameter of 6 cm, fuzzysurface, the margin wavy, translucent whitish in colour

4.25 cm long, vertical, gelatinous, colour similar to cap

Whitish spines running down the stem, fuzzy in touch

Pseudohydnumgelatinosum

Order: Tramellales

Family: Tramellaceae

MBSRJMR1

Globular, 10 cm wide, rough scaly surface, cracked, yellow to brownish in colour

Stalk absent

Purplish black dust like substance “Gleba” are found inside

Scleroderma citrinum

Order: Boletales

Family: Sclerodermatacea

MBSRJ74

7.35 cm wide, semicircular, irregular edges, zones furrowed, properly distinct, brownish

Stubby lateral stalk

Yellowish to brownish circular pores, separated by layers

Ganodermaaustrale

Order: Polyporales

Family: Ganodermataceae

MBSRJ77

6 cm convex undecorated, darker at the centre, margin tuberculate-striate, a darker zone at the inner end of the margin

8.25 cm long, dirtywhite, undecorated withtiny fibrils, hollow, cylindrical, presence of awhite membranous volvalremnant around the stalk

Gills are free, crowded, whitish to creamish in colour

Amanita lignitincta

Order: Agaricales

Family: Amanitaceae

MBSRJ79

7.2 cm plano convex, slimywrinkled, dark brown to reddish brown surface

4.25 cm long, 1 cm thick dotted rough surface, moist gelatinous dots, creamish to dirty white surface

Whitish to yellowish pores covered with white partial veins young

Suillusbrevipes

Order: Boletales

Family: Suillaceae

MBSRJPSI

5.85 cm broadly conical, with a pointed tip at the center, brownish, stratiated margin

Cylindrical hollow stalk of 3.2 cm wide, white in uur, tiny hairy fibrils on the surface

Gills distantly separated, thick beneath the cap attach to the stalk, whitish in colour

Inocybe sp.

Order: Agaricales

Family: Inocybaceae

MBSRJ82

6.45 cm campanulate cap, slightly umbonate, Plano convex, grayish brown darker at the center with tuberculate striated margin

8 cm long, sub-cylindricalwith slightly expanded apex,grayish tiny fibrillose scalesover the stalk surface,incomplete ring like projection at the stalk base

Gills are free, crowded, subtruncate, grayish to somewhat darker at the edges

Amanita sp.

Order: Agaricales

Family: Amanitaceae

MBSRJ83

5.5 cm, flat, brown, striated margin with the presence of adpressed scales, tiny fibrils at the cap surface

6.28 cm long, slender, hollow, ring and volva present with white floccose squamules

Gills are free, crowded, attached to the stalk

Amanita spissacea

Order: Agaricales

Family: Amanitaceae

MBSRJ84

Cap surface of 7.2 cm, white conical to applanate with volval remnants of 0.2 cm wide, tiny fibrilous appendiculate over the surface

Stipe length of 12 cm, attenuated upwards, covered with white floccose squamules with a basal bulb of 3 cm wide, subglobular, with conical warts

Gills are free, attenuated, whitish to creamish in colour

Amanita virgineoides

Order: Agaricales

Family: Amanitaceae

MBSRJ86

5.2 cm flat, convex, greyish brown with darker center, smooth velvety in touch with lined margin

5.5 cm long, tapering at the apex, whitish to light brownish in colour, shaggy with a skirt like ring, enlarge sac like white volva present at the base

Gills are free from the stem, crowded to close, whitish in colour

Amanita spreta

Order: Agaricales

Family: Amanitaceae

Table 3.2

Microscopical analysis of few mushrooms from Meghalaya, India

Mushroom specimen

Spore print

Lactophenol cotton blue staining

Melzer’s staining

Spore morphology

Spore size (μm)

MBSRJ1

Creamy

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 5.8–9.06 × 3.5–4.7

Shape: Cylindrical

Ornamentatioon: Smooth

Mean: 7.43 × 4.1

Mean L/B ratio: 1.81

MBSRJ2

White

Cyanophilic

Amyloid

Symmetry:Symmetrical

Face view: 6.8–7.06 × 5.3–6.23

Shape:Globose

Ornamentation: Spiky warts

Mean: 6.93 × 5.77

Mean L/B ratio: 1.2

MBSRJ6

White

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 8.1–9.76 × 5.5–6.7

Shape:Subglobose

Ornamentation: Smooth

Mean: 8.93 × 6.1

Mean L/B ratio: 1.46

MBSRJ8

Purple brown

Amphophilic

Inamyloid

Symmetry:Symmetrical

Face view: 5.6–7.9 × 3.2–4.7

Shape: Ellipsoidal

Ornamentation: Smooth

Mean: 6.75 × 3.95

Mean L/B ratio: 1.7

MBSRJ11

White

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 6.8–8.06 × 2.8–3.7

Shape:Allantoid

Ornamentation: Smooth

Mean: 7.43 × 3.25

Mean L/B ratio: 2.28

MBSRJ12

White

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 4.8–6.06 × 2.5–4.2

Shape: Ellipsoidal

Ornamentation: Smooth

Mean: 5.43 × 3.35

Mean L/B ratio: 1.62

MBSRJ13

Rusty Brown

Cyanophilic

Inamyloid

Symmetry: Asymmetrical

Face view: 7.8–10.8 × 4.8–6.4

Shape:Subellipsoidal

Mean: 9.3 × 5.6

Ornamentation: Smooth

Mean L/B ratio: 7.45

MBSRJ18

Creamy

Cyanophilic

Amyloid

Symmetry: Symmetrical

Face view: 5.8–7.06 × 5.5–6.7

Shape: Ellipsoidal

Mean: 6.43 × 6.1

Ornamentation: Warts

Mean L/B

ratio: 1.05

MBSRJ26

Creamy

Cyanophilic

Amyloid

Symmetry:Symmetrical

Face view: 6.8–8.06 × 5.5–6.7

Shape:Globose

Ornamentation: Warts

Mean: 7.43 × 6.1

Mean L/B ratio: 1.21

MBSRJ35

Brown

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 10–12.4 × 3.5–4.5

Shape:Subfusoid

Ornamentation: Smooth

Mean: 11.2 × 4

Mean L/B ratio: 3.55

MBSRJ37

White

Cyanophilic

Amyloid

Symmetry:Symmetrical

Face view: 6.8–9.06 × 5.5–5.7

Shape:Subglobose

Ornamentation: Isolated Warts

Mean: 7.93 × 5.6

Mean L/B ratio: 1.41

MBSRJ38

White

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 4.8–5.06 × 2.4–2.7

Shape: Ellipsoidal

Ornamentation: Smooth

Mean: 4.93 x 2.55

Mean L/B ratio: 1.93

MBSRJ39

White

Cyanophilic

Amyloid

Symmetry:Symmetrical

Face view: 7.2–8.06 × 4.5–6.7

Shape: Ellipsoidal

Ornamentation: Isolated warts

Mean: 7.63 x 5.6

Mean L/B ratio: 1.36

MBSRJ45

Greenish brown

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 8.2–8.66 × 3.5–3.7

Shape: Ellipsoidal

Ornamentation: Smooth

Mean: 7.63 × 3.6

Mean L/B ratio: 2.11

MBSRJ48

Whitish buff

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 8.1–9.06 × 3.5–4.0

Shape: Ellipsoidal

Ornamentation: Smooth

Mean: 8.58 × 3.75

Mean L/B ratio: 2.288

MBSRJOY

Creamy

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 6.8–8.1 × 3.5–4.2

Shape: Ellipsoidal

Ornamentation: Smooth

Mean: 7.45 × 3.85

Mean L/B ratio: 1.93

MBSRJ51

Yellow

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 8.8–9.06 × 4.5–5.2

Shape:Fusoid

Ornamentation: Smooth

Mean: 8.93 × 4.85

Mean L/B ratio: 1.84

MBSRJ54

Brownish Yellow

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 9.8–10.1 × 5.5–5.7

Shape:Fusoid

Ornamentation: Smooth

Mean: 9.95 × 5.6

Mean L/B ratio: 1.77

MBSRJ55

White

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 7.8–8.06 × 3.5–3.7

Shape:Fusoid

Ornamentation: Smooth

Mean: 8.94 × 4.59

Mean L/B ratio: 1.94

MBSRJ57

Greenish yellow

Amphophilic

Inamyloid

Symmetry:Symmetrical

Face view: 8.8–10.1 × 3.5–3.7

Shape: Spindle shape

Ornamentation: Smooth

Mean: 9.45 × 3.6

Mean L/B ratio: 2.6

MBSRJ58

Creamy

Cyanophilic

Amyloid

Symmetry:Symmetrical

Face view: 5.8–6.06 × 4.5–5.7

Shape:Globose

Ornamentation: Warts

Mean: 5.93 × 5.1

Mean L/B ratio: 1.16

MBSRJ59

Pale Yellow

Amphophilic

Inamyloid

Symmetry:Symmetrical

Face view: 8.8–9.5 × 2.5–4.7

Shape:Subfusoid

Ornamentation: Smooth

Mean: 9.15 × 3.6

Mean L/B ratio: 2.54

MBSRJ61

Grayish Brown

Amphophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 6.8–7.06 × 5.5–5.7

Shape:Globose

Ornamentation: Ridges and lines forming reticulum

Mean: 6.93–5.6

Mean L/B ratio: 1.23

MBSRJ62

Creamy

Cyanophilic

Amyloid

Symmetry:Symmetrical

Face view: 6.1–6.7 × 4.5–5.7

Shape:Subglobose

Ornamentation: Thick double wall with Warts

Mean: 6.4–5.1

Mean L/B ratio: 1.94

MBSRJ63

Brown

Amphophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 6.8–9.65 × 5.5–5.7

Shape: Ellipsoidal

Ornamentation: Smooth

Mean: 8.22 × 5.6

Mean L/B ratio: 1.46

MBSRJ64

Creamy

Cyanophilic

Amyloid outerwarts

Symmetry:Symmetrical

Face view: 6.8–7.06 × 5.5–5.7

Shape:Subglobose

Ornamentation: Warts

Mean: 6.93 × 5.6

Mean L/B ratio: 1.23

MBSRJGN

Pale creamy

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 6.5–6.8 × 4.5–5.7

Shape:Subglobose

Ornamentation:Smoothe

Mean: 6.65 × 5.1

Mean L/B ratio: 1.3

MBSRJ67

Creamy

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 8.5–10.1 × 3.5–4.1

Shape: Ellipsoidal

Ornamentation:Smooth

Mean: 9.3–3.8

Mean L/B ratio: 2.44

MBSRJ69

White

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 8.3–9.66 × 3.5–3

.7

Shape: Ellipsoidal

Ornamentation:Smooth

Mean: 8.98–3.6

Mean L/B ratio: 2.49

MBSRJ70

White

Cyanophilic

Amyloid

Symmetry:Symmetrical

Face view: 4.5–5.2 × 4.1–4.8

Shape:Subglobose

Ornamentation:Smooth

Mean: 4.85–4.45

Mean L/B ratio: 1.08

MBSRJMR1

Dark brown

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 8–12.06 × 7.5–9.7

Shape:Globose

Ornamentation:Reticulate

Mean: 10.03–8.6

Mean L/B ratio: 1.16

MBSRJ74

Yellow

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 8.8–10.1 × 4.5–5.7

Shape: Ovoid

Ornamentation:Smooth

Mean: 8.94–4.59

Mean L/B ratio: 1.94

MBSRJ77

White

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 9.5–12.5 × 7.5–8.9

Shape:Subglobose

Ornamentation:Smooth

Mean: 11–8.2

Mean L/B ratio: 1.34

MBSRJ79

Yellow

Cyanophilic

Pseudoamyloid

Symmetry:Symmetrical

Face view: 7.8–9.02 × 2.8–3.7

Shape:Subfusoid

Ornamentation:Smooth

Mean: 8.41–3.25

Mean L/B ratio: 2.58

MBSRJPSI

Brown

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 12.8–12.9 × 9.5–10.7

Shape:Fusoid

Ornamentation:Tiny Nodules

Mean: 12.85–10.1

Mean L/B ratio: 1.27

MBSRJ82

White

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 9–12.1 × 8.5–11.8

Shape:Subglobose

Ornamentation:Smooth

Mean: 10.55–10.15

Mean L/B ratio: 1.03

MBSRJ83

White

Cyanophilic

Amyloid

Symmetry:Symmetrical

Face view: 7.8–10.1 × 6.5–9.8

Shape:Fusoid

Ornamentation:Smooth

Mean: 8.95–8.15

Mean L/B ratio: 1.09

MBSRJ84

White

Cyanophilic

Amyloid

Symmetry:Symmetrical

Face view: 9.8–12.1 × 4.5–4.8

Shape: Ellipsoidal to Subglobose

Mean: 10.93–4.65

Ornamentation:Smooth

Mean L/B ratio: 2.35

MBSRJ86

White

Cyanophilic

Inamyloid

Symmetry:Symmetrical

Face view: 7.8–9.5 × 5.5–6.8

Shape: Ellipsoidal

Ornamentation:Smooth

Mean: 8.65–6.15

Mean L/B ratio: 1.4

Epigenetic factors have made the morphological features unstable at both intra- and interspecies level leading to high incongruency thus making the use of molecular marker as a reliable confirmatory identification alternative (Feng et al. 2012). Grouping or characterizing the species based on morphotyping has led to convergent evolution (Brun and Silar 2010). Apart from these, the concepts of exploiting molecular approaches have unraveled the knowledge on the nature of evolution and the phylogenetic concept of the life forms in the last decade. The upgradation of various statistical methods along with bioinformatics tools has aided and simplified the process of evaluation of the evolutionary clade of a species. Approaches such as amplification of specific hypervariable gene loci by polymerase chain reaction (PCR) or restriction digestion of a specific gene sequence using restriction fragment length polymorphism (RFLP) have provided deeper understanding on the typification and diversity of species.

Till recently, the absence of a universally accepted DNA barcode for fungi, the second most specious eukaryotic kingdom (Blackwell 2011; Mora et al. 2011), has been a serious limitation for biodiversity studies. For proper identification and discovery of true cryptic species and classifying them in the pre-existent evolutionary clade in fungi, there is a felt need for developing molecular barcoding tools and markers in mushrooms.

3.2 Paradigm Shift from Classical to Advanced Molecular Approaches

Mycology is mostly familiarized to the lower species of fungi without much attention being paid to describe and identifies cryptic species. Mushrooms studies have been revolving around wood rotten fungi and the poisonous ‘toadstools’. It is well known that fungi are known to be found everywhere presenting morphological similarity with one another thus making the differentiation among the closely related species very difficult. Till the last decade, the gross identification of these cryptic species was solely dependent on the morphotypic features of the individual which were later supplemented with the in vitro micro graphical characters. The advancement in molecular biology tools in the recent era has resulted in shift to a new level of nucleic acid and its sequence analysis leading to nucleic acid revolution in mushroom identification. The levels of genetic information contained at the cellular level are far more advanced than that of the phenotypic features. Several variations observable at the genetic level can be incorporated to the phenotypic features and are collectively considered under “epigenetic” factors.

The taxonomic study in fungi is associated with 80,000 species, traditionally differentiated into six main domain based on the sexual reproductive units, ultrastructural components and biochemical analysis. These domains include Myxomycetes or Protists, Oomycetes and the true fungi which are classified into Ascomycetes, Basidiomycetes, Zygomycetes and Chytrids (McLaughlin et al. McLaughlin et al. 2001a, 2001b). A pseudo-division includes the Deuteromycetes or Fungi Imperfecti commonly referred to as moulds or rust have asexually reproducing unit and some known sexual reproductive stages. With an estimate of 2.2 to 3.8 million fungal species (Hawksworth and Lacking 2017), the problem with cataloguing of fungi in the database has already become a challenging task without the contribution of molecular information.

In the last decades, taxonomist have classified the species based on morphology, niche types, growth behavior, their interaction with the host and to the ecological factors (Fig. 3.2). Thus the classification and identification of the cryptic species from the taxonomical point of view is far from being complete. It is well accepted that less than 1% of the microbiological species are culturable in in-vitro. So, the need of the biological markers for easy identification of the unculturable environmental samples are of utmost importance developing barcodes for forms like mushrooms is expected to answer the difficult questions posed in their classification approaches.
Fig. 3.2

Percentage of using various markers for the identification of the fungal species (Adapted from Raja et al. 2017)

3.3 Molecular Markers for Identification

With the advancement of the barcode in the prokaryotic system, amplification of the nucleic acids and the initial construction of the phylogenetic map work on fungi began with nuclear ribosomal genes. The structural gene sequence of rRNA is known for its well conserved regions at both genus and species level. Initially 18SrRNA , a homolog to 16S rRNA in bacteria was used for the phylogenetic analysis but the region has limitations of having fewer hypervariable domains. The large ribosomal subunit 28S rRNA gene discriminates the species on its own or combines with the ITS region. The internal transcribed spacer region (ITS) of the ribosomal region of DNA (rDNA) is commonly used as a marker for resolution at or below the genus level (Seifert 2009; Begerow et al. 2010) and is a recommended marker by the International Fungal Barcoding Consortium (Schoch et al. 2012). In fungi, the ITS ranges from 550–600 bp comprising of two hypervariable spacer regions, ITS1 and ITS2 which are separated by highly conserved 5.8S rRNA gene (White et al. 1990). The region is flanked by 18S rRNA and 28S rRNA genes towards the 5′-end and 3′-end of ITS-1 and ITS-2 spacer region respectively. In fungi, ITS is found in multiple tandem copies in each haploid genome making it highly desirable for use as a fungal barcode aided by its easier amplification even in trace amounts of biological samples and is extensively used for the genetic distance mapping in fungi. ITS has been used as a fungal universal barcode for the identification of the cryptic species and has been cited in more than 500 literature (Schoch et al. 2012). Apart from the ITS, the small ribosomal subunit (SSU) region when combined with the NS1 and NS4 region gives an information about the phylogenetic placement of higher fungi in taxonomical level. Similarly for the identification of the species at the intermediate taxonomical level, large ribosomal subunit (LSU) primers in combination with LROR and LR6 are being used (Vilgalys and Hester 1990). These genes are widely used as markers in the fungal systematic nomenclature such as Assembling the Fungal Tree of Life (AFTOL).

In mycological identification, the sequence obtained for a species of fungi is compared with the unknown sequence from database available in Sequence Database such as GenBank at NCBI (National Centre for Biotechnology Information) and EMBL, The European Nucleotide Sequence Archive of European Molecular Biology Laboratory.

Apart from the ITS region, the mitochondrial gene, which codes for the cytochrome oxidase I (COX I) used as a biomarker in the animal kingdom have been explored for fungal systems but are found to be inappropriate for the identification at species level for fungi (Schoch et al. 2012). The protein encoding genes like RNA polymerase II (RPB1, RPB2) are being widely used for the phylogenetic analysis of fungi but due to it ubiquitous and single copy number and slow rate of sequence diversion it is not highly recommended (Tanabe et al. 2002). For species level identification of fungi in general, the short ribosomal subunit (SSU) has no such barcode gap and is not recommended as an identification marker. The large ribosomal subunit (LSU) sometimes show no such amplification in diverse species adding to editing error and are not considered at par with the usage of SSU marker. However, the hypervariable ITS regions have much superior barcode gap and due to its tandem repeats and resolving power to distinguish the mushrooms at its species level, it is a defined barcode for the phylogenetic analysis of the mushrooms at its basic level of species discrimination phylogenetically.

3.4 ITS Marker Assisted Identification of Mushrooms

Polyphasic characterization gives a preliminary identification of a species but genus level identification is always left to a dilemma when considering the macroscopic and microscopic features. Few mushroom species look morphologically alike to other species as observed in a study carried to characterize wild mushrooms by the authors` group. For example, the specimen MBSRJ1and MBSRJ11 is a common wood fungus alike to Trametes species but identification based on the molecular marker using ITS identified the specimens as Formitopsis ostreoformis and Trichaptum biforme respectively (Fig. 3.3). In the same study, the morphological identification of few mushroom species could not be completed due to the lack of information and relevant data in the existing databases. The paucity in the morphological data can also be highly affected by the epigenetic factors. The mushroom species MBSRJ18 and MBSRJ39 were morphologically alike but confirming the molecular data resembled them as two different species, Lactarius glaucescens and Lactifluus vellereus respectively. The molecular identification using the ITS loci was first time reported in the study for MBSRJ82 which was identified to be Amanita griseofolia. The morphological trait in these cryptic species has always leaded to misidentification which may be the effect of convergent evolution. Thus confirming the use of molecular marker such as ITS for correct identification is necessary along with the traditional technique of identifying the mushrooms. Hence employing the phylogenetic analyses becomes a useful tool for classification of unknown sequence and incorporating such emerging information into the existing evolutionary framework.
Fig. 3.3

Evolutionary distance relationship of few mushroom species based on ITS sequence similarity

3.5 Conclusion

Though a large number of the universal biomarkers for the phylogenetic analysis of fungi in general and macrofungi in particular have been in use, the markers are not the only restricted as genetic markers in phylogenetic analysis. There is a need for exploration of additional biomarkers for fungal taxonomy . Compared to the earlier approaches of identifying the fungal species with the basic morphological and microscopic features, there is a need towards improving the molecular markers along with the algorithms of various statistical tools which can provide an appropriate authentication for the identification of the fungi. The combination of both morphotyping and molecular genetic marker studies will relatively assure a full proof system for the phylogenetic analysis of the higher eukaryotes like mushrooms. The need for biomarker is not only essential to delineate the existing morphotyping but also to authenticate and classify the novel undescribed forms of macrofungi by linking them with available sequences in various databases and genebanks.

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Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Microbiology Laboratory, Department of Biotechnology & BioinformaticsNorth-Eastern Hill UniversityShillongIndia

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